Conjugates of hydroxyalkyl starch and a protein, prepared by native chemical ligation

ABSTRACT

Conjugates of an active substance and hydroxyalkyl starch (HAS) are provided herein. The active substance and the HAS are linked by a chemical moiety having a structure according to formula (I) wherein Y is O or S, and X is SH or (F). The conjugate has a structure according to formula (IV) wherein HAS′ is a residue of HAS or a derivative thereof linked to the thioester group, and AS′ is a residue of the active substance or derivative thereof linked to the alpha-X beta-amino group; or a structure according to formula (V) wherein HAS′ is a residue of HAS or derivative thereof linked to the alpha-X beta-amino group, and AS′ is a residue of the active substance or derivative thereof linked to the thioester group, and wherein the group —(C═Y) is derived from the thioester group —(C═Y)—S—R′ and the group HN—CH—CH 2 —X is derived from the alpha-X beta amino group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and claims benefit under 35U.S.C. §119(a) of International Application No. PCT/EP2005/002640,having an International Filing Date of Mar. 11, 2005, which published inEnglish as International Publication Number WO 2005/092391, and whichclaims the benefit of priority of U.S. Provisional Application Ser. No.60/552,069, filed on Mar. 11, 2004, and European Application Serial No.04005875.2, filed on Mar. 11, 2004.

TECHNICAL FIELD

The present invention relates to conjugates of an active substance andhydroxyalkyl starch, wherein the conjugates are prepared by nativechemical ligation. The present invention also relates to the method ofproducing these conjugates and the use of the conjugates.

BACKGROUND

Native chemical ligation is a highly effective method for preparinglarge peptides and small proteins where a peptide thioester is coupledwith a peptide bearing an N-terminal cysteine residue to provide aproduct containing an amide bond at the site of the ligation.

WO 03/031581 A2 discloses a method of conjugating a polymer derivativeto a polypeptide having a cysteine or histidine residue at theN-terminus, wherein said method comprises providing a polypeptide havinga cysteine or histidine residue at the N-terminus, providing athioester-terminated polymer, the polymer comprising a water soluble andnon-peptidic polymer backbone, preferably a polyethylene glycolepolymer, and reacting the polymer derivative and the polypeptide. Aspolymers, poly(alkylene glycol), copolymers of ethylene glycol andpropylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(alpha hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),polyacrylate, polyacrylamides, polysaccharides, and copolymers,terpolymers and mixtures thereof. All explicitly disclosed polymers arepolyethylenglycol polymers.

Despite the progress of coupling methods and use of monofunctionalPEG-molecules, a general disadvantage of PEGylated drugs is that themetabolization pathway of PEG as a non-natural polymer is not known indetail.

SUMMARY

Therefore, it was an object of the present invention to provide novelconjugates of an active substance and a polymer formed by chemicalligation wherein no polyalkylene glycol, especially no polyethyleneglycol is used as polymer.

Accordingly, it was another object of the present invention to provide amethod for preparing these conjugates.

Thus, the present invention relates to a conjugate of an activesubstance and hydroxyalkyl starch, wherein the active substance and thehydroxyalkyl starch are linked by a chemical residue according toformula (I)

wherein Y is a heteroatom, selected from the group consisting of O andS, and wherein X is selected from the group consisting of —SH and

In one aspect, this document features a method for preparing a conjugateof an active substance and hydroxyalkyl starch, wherein the activesubstance and the hydroxyalkyl starch are covalently linked by achemical residue having a structure according to formula

wherein Y is a heteroatom, selected from the group consisting of O andS, the method comprising

-   (i) reacting a thioester group —(C═Y)—S—R′ of a hydroxyalkyl starch    derivative comprising the thioester group with an alpha-X beta amino    group

of an active substance derivative comprising the alpha-X beta aminogroup, or (ii) reacting a thioestergroup —(C═Y)—S—R′ of an activesubstance derivative comprising the thioester group with an alpha-X betaamino group

of a hydroxyalkyl starch derivative comprising the alpha-X beta aminogroup, wherein R′ is selected from the group consisting of hydrogen, anoptionally suitably substituted, linear, cyclic and/or branched alkyl,aryl, heteroaryl, aralkyl, and heteroaralkyl group, preferably benzyl,wherein X is selected from the group consisting of SH and

and wherein the group —(C═Y) is derived from the thioester group—(C═Y)—S—R′ and the group HN—CH—CH₂—X is derived from the alpha-X betaamino group. A thioester functionalized hydroxyalkyl starch can bereacted with an alpha-X beta-amino group of the active substance whereinthe alpha-X beta-amino group is comprised in a cysteine or histidineresidue of the active substance. The method can include oxidizinghydroxyalkyl starch at its reducing end and (i) converting the oxidizedreducing end to an activated carboxylic acid derivative and reacting theactivated carboxylic acid derivative with a compound R′-SH; or (ii)reacting the oxidized reducing end with a carbodiimide and a thiol R′SHto give the thioester functionalized hydroxyalkyl starch. The method caninclude oxidizing hydroxyalkyl starch at its reducing end, reacting theoxidized reducing end with an at least bifunctional compound comprisingtwo amino groups to give an amino functionalized hydroxyalkyl starchderivative, and reacting the amino group of the derivative with an atleast bifunctional compound comprising at least one functional groupwhich is reacted with the amino group of the derivative, and comprisingat least one thioester group.

A thioester functionalized active substance can be reacted with analpha-X beta-amino group of a hydroxyalkyl starch derivative, and themethod can include oxidizing hydroxyalkyl starch at its reducing end andreacting the oxidized reducing end with a functional group Z of acompound comprising, in addition to Z, an alpha-X beta-amino group. Thecompound comprising Z and the alpha-X beta-amino group can be1,3-diamino-2-thio propane or 2,3-diamino-1-thio propane.

A thioester functionalized active substance can be reacted with analpha-X beta-amino group of a hydroxyalkyl starch derivative, and themethod can include oxidizing hydroxyalkyl starch at its reducing end,reacting the oxidized reducing end with a functional group Z of acompound comprising, in addition to Z, a further functional group W, togive a first hydroxyalkyl starch derivative, and reacting the functionalgroup W of the first hydroxyalkyl starch derivative with a functionalgroup V of a compound comprising, in addition to V, an alpha-Xbeta-amino group, to give the alpha-X beta-amino functionalizedhydroxyalkyl starch derivative. The compound comprising Z and W can be adiamino functionalized compound. The compound comprising V and thealpha-X beta-amino can be cysteine or a derivative thereof or histidineor a derivative thereof, V being the carboxy group or a reactive carboxygroup, preferably a reactive ester or a carboxylic acid anhydride. Theactive substance can be a small molecule drug comprising a carboxygroup, and the method can include (i) converting the carboxy group to anacid chloride with a compound R′-SH via alkylthio-dehalogenation, or(ii) reacting the carboxy group with a carbodiimide and a thiol R′SH togive the thioester functionalized active substance. The active substancecan be a peptide which was produced using a synthesis resin allowing fora thioester functionalized peptide. The active substance can be aprotein which was produced using an expression vector leading to athioester functionalized protein.

In another aspect, this document features a conjugate of an activesubstance and hydroxyalkyl starch, as obtainable by a method describedherein.

In another aspect, this document features a conjugate of an activesubstance and hydroxyalkyl starch, wherein the active substance and thehydroxyalkyl starch are linked by a chemical moiety, having a structureaccording to formula

wherein Y is a heteroatom, selected from the group consisting of O andS, and X is selected from the group consisting of SH and

the conjugate having a structure according to formula

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the thioester group, and wherein AS′ is theresidue of the active substance or a derivative thereof which was linkedto the alpha-X beta-amino group, or a structure according to formula

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the alpha-X beta-amino group, and whereinAS′ is the residue of the active substance or a derivative thereof whichwas linked to the thioester group, and wherein the group —(C═Y) isderived from the thioester group —(C═Y)—S—R′ and the group HN—CH—CH₂—Xis derived from the alpha-X beta amino group. The conjugate can have astructure according to formula

-   -   or according to formula

-   -   or according to formula

-   wherein n=0 or 1,-   wherein L is an optionally suitably substituted, linear, cyclic    and/or branched alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl    residue with 2 to 10 carbon atoms in the respective alkyl residue,    wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkyl    group, preferably a hydroxyethyl group, and wherein HAS″ refers to    the HAS molecule without the terminal saccharide unit at the    reducing end. The active substance can be selected from the group    consisting of proteins, peptides, and small molecule drugs. The    hydroxyalkyl starch can be hydroxyethyl starch having a mean    molecular weight of from 1 to 30010, preferably from 2 to 20010,    more preferably from 4 to 130 kD; and a degree of substitution in    the range of from about 0.1 to about 0.8 (e.g., from 0.1 to 0.8).

In another aspect, this document features an alpha-X beta-aminofimctionalized hydroxyalkyl starch derivative according to formula

-   -   or according to formula

-   -   or according to formula

wherein L is an optionally suitably substituted, linear, cyclic and/orbranched alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl residue with2 to 10 carbon atoms in the respective alkyl residue, wherein HAS″refers to the HAS molecule without the terminal saccharide unit at thereducing end, and wherein R₁, R₂ and R₃ are independently hydrogen or ahydroxyalkyl group, preferably a hydroxyethyl group, and wherein X isselected from the group consisting of SH and

wherein HAS″ refers to the HAS molecule without the terminal saccharideunit at the reducing end.

In still another aspect, this document features a thioesterfunctionalized hydroxyalkyl starch derivative according to formulaaccording to formula

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, preferably a hydroxyethyl group, wherein HAS″ refers to the HASmolecule without the terminal saccharide unit at the reducing end, andwherein S—R′ is an electrophilic leaving group, preferably selected fromthe group consisting of substituted or unsubstituted thiophenol,thiopyridine, benzyl mercaptane, ethanethiol, methanethiol,2-mercaptoethansulfonic acid, 2-mercaptoacetic acid, 2-mercaptoaceticacid methyl or ethyl ester, 3-mercaptopropionic acid,3-mercaptopropionic acid methyl or ethyl ester, 4-mercaptobutyric acid,and 4-mercaptobutyric acid methyl or ethyl ester.

This document also features a thioester functionalized hydroxyalkylstarch derivative according to formula according to formula

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, preferably a hydroxyethyl group, wherein HAS″ refers to the HASmolecule without the terminal saccharide unit at the reducing end, andwherein S—R′ is an electrophilic leaving group, preferably selected fromthe group consisting of substituted or unsubstituted thiophenol,thiopyridine, benzyl mercaptane, ethanethiol, methanethiol,2-mercaptoethansulfonic acid, 2-mercaptoacetic acid, 2-mercaptoaceticacid methyl or ethyl ester, 3-mercaptopropionic acid,3-mercaptopropionic acid methyl or ethyl ester, 4-mercaptobutyric acid,and 4-mercaptobutyric acid methyl or ethyl ester, wherein wherein L₁ andL₂ are independently an optionally substituted, linear, branched and/orcyclic hydrocarbon residue, optionally comprising at least oneheteroatom, comprising an alkyl, aryl, aralkyl, heteroalkyl, and/orheteroaralkyl moiety, the residue having from 1 to 60 preferably from 1to 40, more preferably from 1 to 20, more preferably from 1 to 10 carbonatoms, and wherein D is a linkage, preferably a covalent linkage whichwas formed by a suitable functional group F₂ linked to L₁, and asuitable functional group F₃ linked to L₂, and wherein F₃ is capable offorming a chemical linkage with F₂. L₁ and L₂ can be independently—(CH₂)_(n)— with n=2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2, 3, 4, 5,6, more preferably 2, 3, 4, and especially preferably 4.

F₂ and F₃ can independently be selected from the group consisting of

-   -   a C—C-double bond or C—C-triple bond or aromatic C—C-bond;    -   a thio group or a hydroxy group;    -   an alkyl sulfonic acid hydrazide, or an aryl sulfonic acid        hydrazide;    -   a 1,2-diol;    -   a 1,2 amino-thioalcohol;    -   an azide;    -   a 1,2-aminoalcohol;    -   an amino group —NH₂ or a derivative of the an amino group        comprising the structure unit —NH— such as aminoalkyl groups,        aminoaryl group, aminoaralkyl groups, or alkarlyamino groups;    -   a hydroxylamino group —O—NH₂, or derivative of a hydroxylamino        group comprising the structure unit —O—NH—, such as        hydroxylalkylamino groups, hydroxylarylamino groups,        hydroxylaralkylamino groups, or hydroxalalkarylamino groups;    -   an alkoxyamino group, aryloxyamino group, aralkyloxyamino group,        or alkaryloxyamino group, each comprising the structure unit        —NH—O—;    -   a residue having a carbonyl group, -Q-C(=G)-M, wherein G is O or        S, and M is, for example,        -   —OH or —SH;        -   an alkoxy group, an aryloxy group, an aralkyloxy group, or            an alkaryloxy group;        -   an alkylthio group, an arylthio group, an aralkylthio group,            or an alkarylthio group;        -   an alkylcarbonyloxy group, an arylcarbonyloxy group, an            aralkylcarbonyloxy group, or an alkarylcarbonyloxy group;        -   an activated ester (e.g., an ester of a hydroxylamine)            having imide structure such as N-hydroxysuccinimide or            having a structure unit O—N where N is part of a heteroaryl            compound or, with G=O and Q absent, such as aryloxy            compounds with a substituted aryl residue such as            pentafluorophenyl, paranitrophenyl or trichlorophenyl an            aryloxy compound with a substituted aryl residue;

wherein Q is absent or NH or a heteroatom such as S or O;

-   -   —NH—NH₂, or —NH—NH—;    -   —NO₂;    -   a nitril group;    -   a carbonyl group such as an aldehyde group or a keto group;    -   a carboxy group;    -   a —N═C═O group or a —N═S group;    -   a vinyl halide group such as vinyl iodide or vinyl bromide or a        triflate group;    -   —C≡C—H;    -   —(C═NH₂Cl)—OAlkyl    -   a —(C═O)—CH₂-Hal group wherein Hal is Cl, Br, or I;    -   —CH═CH—SO₂—;    -   a disulfide group comprising the structure —S—S—;    -   the group

and

-   -   the group

and wherein F₃ is a functional group capable of forming a chemicallinkage with F₂ and is preferably selected from the above-mentionedgroups, F₂ preferably comprising the moiety —NH—, more preferablycomprising an amino group, F₃ preferably comprising the moiety —(C=G)—,more preferably-(C═O)—, more preferably the moiety-(C)-G-, still morepreferably-(C═O)-G-, and especially preferably-(C═O)—O, D beingparticularly preferably an amide linkage.

In yet another aspect, this document features a method for the treatmentof a human or animal body. The method can include administering aconjugate described herein to a human or animal in need of treatment.

In another aspect, this document features a pharmaceutical compositioncomprising a conjugate as described herein. The pharmaceuticalcomposition can further include at least one pharmaceutically acceptablediluent, adjuvant, or carrier.

In another aspect, this document features a composition comprising aconjugate of an active substance and hydroxyalkyl starch as describedherein. This document also features a composition comprising afunctionalized hydroxyalkyl starch derivative (e.g., an alpha-Xbeta-amino functionalized hydroxyalkyl starch derivative) as describedherein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows an analysis of the crude peptide-thioester Cys-HESconjugates according to example 2.1, by gel electrophoresis. A XCellSure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143power supply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris geltogether with a MES SDS running buffer at reducing conditions (bothInvitrogen GmbH, Karlsruhe, D) were used according to the manufacturer'sinstruction.

Lane A: Roti®-Mark STANDARD (Carl Roth GmbH+Co. KG, Karlsruhe, D)Molecular weight marker from top to bottom: 200 KD, 119 KD, 66 KD, 43KD, 29 KD, 20 KD, 14.3 KD;

Lane B: Conjugation of Peptide-Thioester A to H-Cys(S-tBu)-HES10/0.4employing thiophenol as catalyst;

Lane C: Conjugation of Peptide-Thioester A to H-Cys(S-tBu)-HES10/0.4employing benzyl mercaptan as catalyst;

Lane D: Ron®-Mark STANDARD;

Lane E: Conjugation of Peptide-Thioester A to H-Cys(S-tBu)-HES10/0.4without catalyst.

FIG. 2 shows an analysis of the crude Cys-peptide thioester-HESconjugates according to example 2.2 by gel electrophoresis. A XCell SureLock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 powersupply (CONSORTnv, Turnhout, B) were employed. A 12% Bis-Tris geltogether with a MES SDS running buffer at reducing conditions (bothInvitrogen GmbH, Karlsruhe, D) were used according to the manufacturer'sinstruction.

Lane A: Roti®-Mark STANDARD (Carl Roth GmbH+Co. KG, Karlsruhe, D)Molecular weight marker from top to bottom: 200 KD, 119 KD, 66 KD, 43KD, 29 KD, 20 KD, 14.3 KD;

Lane B: Conjugation of Thioester-HES10/0.4 to GM-CSF Inhibitor Peptide(54-78) employing thiophenol as catalyst;

Lane C: GM-CSF Inhibitor Peptide (54-78).

FIG. 3 shows an analysis of the crude EPO thioester-HES conjugatesaccording to example 2.3 by gel electrophoresis. A XCell Sure Lock MiniCell (Invitrogen GmbH, Karlsruhe, D) and a Consort E143 power supply(CONSORTnv, Turnhout, B) were employed. A 10% Bis-Tris gel together witha MOPS SDS running buffer at reducing conditions (both Invitrogen GmbH,Karlsruhe, D) were used according to the manufactures instruction.

Lane A: Roti®-Mark STANDARD (Carl Roth GmbH+Co. KG, Karlsruhe, D)Molecular weight marker from top to bottom: 200 KD, 119 KD, 88 KD, 43KD, 29 KD, 20 KD, 14.3 KD;

Lane B: Conjugation of 6.9 equiv. thioester-HES to EPO employingthiophenol as catalyst;

Lane C: Conjugation of 34.5 equiv. thioester-HES to EPO employingthiophenol as catalyst;

Lane D: EPO without thioester-HES, employing thiophenol as catalyst.

DETAILED DESCRIPTION

Therefore, the term “alpha-X beta-amino group” as used in the context ofthe present invention refers to an ethylene group in which X is bondedto a carbon atom and a primary amino group is bonded to the neighbouringcarbon atom.

In the chemical moiety according to formula (I) above, the group —(C═Y)is derived from the thioester group —(C═Y)—S—R′ and the groupHN—CH—CH₂—X is derived from the alpha-X beta amino group.

The term “active substance” as used in the context of the presentinvention relates to a substance which can affect any physical orbiochemical property of a biological organism including, but not limitedto, viruses, bacteria, fungi, plants, animals, and humans. Inparticular, the term “active substance” as used in the context of thepresent invention relates to a substance intended for diagnosis, curemitigation, treatment, or prevention of disease in humans or animals, orto otherwise enhance physical or mental well-being of humans or animals.Examples of biologically active molecules include, but are not limitedto, peptides, proteins, enzymes, small molecule drugs, dyes, lipids,nucleosides, oligonucleotides, polynucleotides, nucleic acids, cells,viruses, liposomes, microparticles, and micelles. Examples of proteinsinclude, but are not limited to, erythropoietin (EPO) such asrecombinant human EPO (rhEPO), colony-stimulating factors (CSF), such asG-CSF like recombinant human G-CSF (rhG-CSF), alpha-Interferon (IFNalpha), beta-Interferon (IFN beta) or gamma-Interferon (IFN gamma), suchas IFN alpha and IFN beta like recombinant human IFN alpha or IFN beta(rhIFN alpha or rhIFN beta), interleukines, e.g. IL-1 to IL-18 such asIL-2 or IL-3 like recombinant human IL-2 or IL-3 (rhIL-2 or rhIL-3),serum proteins such as coagulation factors II-XIII like factor VIII,alphal-antitrypsin (A1AT), activated protein C (APC), plasminogenactivators such as tissue-type plasminogen activator (tPA), such ashuman tissue plasminogen activator (hTPA), AT III such as recombinanthuman AT III (rhAT III), myoglobin, albumin such as bovine serum albumin(BSA), growth factors, such as epidermal growth factor (EGF),thrombocyte growth factor (PDGF), fibroblast growth factor (FGF),brain-derived growth factor (BDGF), nerve growth factor (NGF), B-cellgrowth factor (BCGF), brain-derived neurotrophic growth factor (BDNF),ciliary neurotrophic factor (CNTF), transforming growth factors such asTGF alpha or TGF beta, BMP (bone morphogenic proteins), growth hormonessuch as human growth hormone, tumor necrosis factors such as TNF alphaor TNF beta, somatostatine, somatotropine, somatomedines, hemoglobin,hormones or prohormones such as insulin, gonadotropin,melanocyte-stimulating hormone (alpha-MSH), triptorelin, hypthalamichormones such as antidiuretic hormones (ADH and oxytocin as well asreleasing hormones and release-inhibiting hormones, parathyroid hormone,thyroid hormones such as thyroxine, thyrotropin, thyroliberin,prolactin, calcitonin, glucagon, glucagon-like peptides (GLP-1, GLP-2etc.), exendines such as exendin-4, leptin, vasopressin, gastrin,secretin, integrins, glycoprotein hormones (e.g. LH, FSH etc.),melanoside-stimulating hormones, lipoproteins and apo-lipoproteins suchas apo-B, apo-E, apo-L_(a), immunoglobulins such as IgG, IgE, IgM, IgA,IgD and fragments thereof, hirudin, tissue-pathway inhibitor, plantproteins such as lectin or ricin, bee-venom, snake-venom, immunotoxins,antigen E, alpha-proteinase inhibitor, ragweed allergen, melanin,oligolysine proteins, RGD proteins or optionally corresponding receptorsfor one of these proteins; or a functional derivative or fragment of anyof these proteins or receptors. Examples of enzymes include, but are notlimited to, carbohydrate-specific enzymes, proteolytic enzymes,oxidases, oxidoreductases, transferases, hydrolases, lyases, isomerases,kinases and ligases. Specific non-limiting examples are asparaginase,arginase, arginin deaminase, adenosin deaminase, glutaminase,glutaminase-asparaginase, phenylalanin, tryptophanase, tyrosinase,superoxide dismutase (SOD), endotoxinase, catalase, peroxidase,kallikrein, trypsin, chymotrypsin, elastase, thermolysin, lipase,uricase, adenosine diphosphatase, purine nucleoside phosphorylase,bilirubin oxidase, glucose oxidase, glucodase, gluconate oxidase,galactosidase, glucocerebrosidase, glucuronidase, hyaluronidase, tissuefactor, streptokinase, urokinase, MAP-kinases, DNAses, RNAses,lactoferrin and functional derivatives or fragments thereof.

According to one alternative of the present invention, the activesubstance is a small molecule drug, a peptide, and/or a protein.

Among others, the following proteins are to be mentioned explicitly:erythropoietin (EPO) such as recombinant human EPO (rhEPO),colony-stimulating factors (CSF), such as G-CSF like recombinant humanG-CSF (rhG-CSF), serum proteins such as coagulation factors II-XIII likefactor VII, factor VIII, or factor IX, alpha1-antitrypsin (A1AT),activated protein C (APC), plasminogen activators such as tissue-typeplasminogen activator (tPA), such as human tissue plasminogen activator(hTPA), AT III such as recombinant human AT III (rhAT III).

In the context of the present invention, the term “hydroxyalkyl starch”(HAS) refers to a starch derivative which has been substituted by atleast one hydroxyalkyl group. A preferred hydroxyalkyl starch of thepresent invention has a constitution according to formula (II)

wherein the reducing end of the starch molecule is shown in thenon-oxidized form and the terminal saccharide unit of HAS is shown inthe acetal form which, depending on e.g. the solvent, may be inequilibrium with the aldehyde form. The abbreviation HAS″ as used in thecontext of the present invention refers to the HAS molecule without theterminal saccharide unit at the reducing end of the HAS molecule.

The term hydroxyalkyl starch as used in the present invention is notlimited to compounds where the terminal carbohydrate moiety compriseshydroxyalkyl groups R₁, R₂, and/or R₃ as depicted, for the sake ofbrevity, in formula (II), but also refers to compounds in which at leastone hydroxyalkyl group is present anywhere, either in the terminalcarbohydrate moiety and/or in the remaining part of the starch molecule,HAS″, is substituted by a hydroxyalkyl group R₁, R₂, or R₃.

Hydroxyalkyl starch comprising two or more different hydroxyalkyl groupsare also possible.

The at least one hydroxyalkyl group comprised in HAS may contain two ormore hydroxy groups. According to a preferred embodiment, the at leastone hydroxyalkyl group comprised HAS contains one hydroxy group.

The expression “hydroxyalkyl starch” also includes derivatives whereinthe alkyl group is mono- or polysubstituted. In this context, it ispreferred that the alkyl group is substituted with a halogen, especiallyfluorine, or with an aryl group. Furthermore, the hydroxy group of ahydroxyalkyl group may be esterified or etherified.

Furthermore, instead of alkyl, also linear or branched substituted orunsubstituted alkene groups may be used.

Hydroxyalkyl starch is an ether derivative of starch. Besides of saidether derivatives, also other starch derivatives can be used in thecontext of the present invention. For example, derivatives are usefulwhich comprise esterified hydroxy groups. These derivatives may be e.g.derivatives of unsubstituted mono- or dicarboxylic acids with 2-12carbon atoms or of substituted derivatives thereof. Especially usefulare derivatives of unsubstituted monocarboxylic acids with 2-6 carbonatoms, especially derivatives of acetic acid. In this context, acetylstarch, butyryl starch and propionyl starch are preferred.

Furthermore, derivatives of unsubstituted dicarboxylic acids with 2-6carbon atoms are preferred.

In the case of derivatives of dicarboxylic acids, it is useful that thesecond carboxy group of the dicarboxylic acid is also esterified.Furthermore, derivatives of monoalkyl esters of dicarboxylic acids arealso suitable in the context of the present invention.

For the substituted mono- or dicarboxylic acids, the substitute groupsmay be preferably the same as mentioned above for substituted alkylresidues.

Techniques for the esterification of starch are known in the art (seee.g. Klemm D. et al, Comprehensive Cellulose Chemistry Vol. 2, 1998,Whiley-VCH, Weinheim, N.Y., especially chapter 4.4, Esterification ofCellulose (ISBN 3-527-29489-9).

According to a preferred embodiment of the present invention,hydroxyalkyl starch according to above-mentioned formula (II) isemployed. The other saccharide ring structures comprised in HAS″ may bethe same as or different from the explicitly described saccharide ring.

As far as the residues R₁, R₂ and R₃ according to formula (II) areconcerned there are no specific limitations. According to a preferredembodiment, R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, a hydroxyaryl group, a hydroxyaralkyl group or a hydroxyalkarylgroup having of from 2 to 10 carbon atoms in the respective alkylresidue or a group (CH₂CH₂O)_(n)—H, wherein n is an integer, preferably1, 2, 3, 4, 5, or 6. Hydrogen and hydroxyalkyl groups having of from 2to 10 are preferred. More preferably, the hydroxyalkyl group has from 2to 6 carbon atoms, more preferably from 2 to 4 carbon atoms, and evenmore preferably from 2 to 4 carbon atoms. “Hydroxyalkyl starch”therefore preferably comprises hydroxyethyl starch, hydroxypropyl starchand hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropylstarch are particularly preferred and hydroxyethyl starch is mostpreferred.

The alkyl, aryl, aralkyl and/or al caryl group may be linear or branchedand suitably substituted.

Therefore, the present invention also relates to a method an a conjugateas described above wherein R₁, R₂ and R₃ are independently hydrogen or alinear or branched hydroxyalkyl group with from 2 to 6 carbon atoms.

Thus, R₁, R₂ and R₃ preferably may be hydroxyhexyl, hydroxypentyl,hydroxybutyl, hydroxypropyl such as 2-hydroxypropyl, 3-hydroxypropyl,2-hydroxyisopropyl, hydroxyethyl such as 2-hydroxyethyl, hydrogen andthe 2-hydroxyethyl group being especially preferred.

Therefore, the present invention also relates to a method and aconjugate as described above wherein R₁, R₂ and R₃ are independentlyhydrogen or a 2-hydroxyethyl group, an embodiment wherein at least oneresidue R₁, R₂ and R₃ being 2-hydroxyethyl being especially preferred.

Hydroxyethyl starch (HES) is most preferred for all embodiments of thepresent invention.

Therefore, the present invention relates to the method and the conjugateas described above, wherein the polymer is hydroxyethyl starch and thepolymer derivative is a hydroxyethyl starch derivative.

Hydroxyethyl starch (HES) is a derivative of naturally occurringamylopectin and is degraded by alpha-amylase in the body. HES is asubstituted derivative of the carbohydrate polymer amylopectin, which ispresent in corn starch at a concentration of up to 95% by weight. HESexhibits advantageous biological properties and is used as a bloodvolume replacement agent and in hemodilution therapy in the clinics(Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; andWeidler et al., 1991, Arzneim.-Forschung/Drug Res., 41, 494-498).

Amylopectin consists of glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physico-chemical properties ofthis molecule are mainly determined by the type of glycosidic bonds. Dueto the nicked alpha-1,4-glycosidic bond, helical structures with aboutsix glucose-monomers per turn are produced. The physico-chemical as wellas the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. There are two possibilities of describing thesubstitution degree:

-   1. The degree of substitution can be described relatively to the    portion of substituted glucose monomers with respect to all glucose    moieties.-   2. The degree of substitution can be described as the molar    substitution, wherein the number of hydroxyethyl groups per glucose    moiety are described.

In the context of the present invention, the degree of substitution,denoted as DS, relates to the molar substitution, as described above(see also Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278,as cited above, in particular p. 273).

HES solutions are present as polydisperse compositions, wherein eachmolecule differs from the other with respect to the polymerisationdegree, the number and pattern of branching sites, and the substitutionpattern. HES is therefore a mixture of compounds with differentmolecular weight. Consequently, a particular HES solution is determinedby average molecular weight with the help of statistical means. In thiscontext, M_(n) is calculated as the arithmetic mean depending on thenumber of molecules. Alternatively, M_(w) (or MW), the weight averagemolecular weight, represents a unit which depends on the mass of theHES.

In the context of the present invention, hydroxyethyl starch maypreferably have a mean molecular weight (weight average molecularweight) of from 1 to 300 kD. Hydroxyethyl starch can further exhibit apreferred molar degree of substitution of from 0.1 to 0.8 and apreferred ratio between C₂:C₆ substitution in the range of from 2 to 20with respect to the hydroxyethyl groups.

The term “mean molecular weight” as used in the context of the presentinvention relates to the weight as determined according to theLALLS-(low angle laser light scattering)-GPC method as described inSommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; andWeidler et al., 1991, Arzneim.-Forschung/Drug Res., 41, 494-498. Formean molecular weights of 10 kD and smaller, additionally, thecalibration was carried out with a standard which had previously beenqualified by LALLS-GPC.

According to a preferred embodiment of the present invention, the meanmolecular weight of hydroxyethyl starch employed is from 1 to 300 kD,more preferably from 2 to 20010, more preferably of from 4 to 130 kD,more preferably from 4 to 100 kD and still more preferably of from 4 to70 kD.

Therefore, the present invention also relates to a method and toconjugates as described above wherein the hydroxyalkyl starch ishydroxyethyl starch having a mean molecular weight of from 4 to 70 kD.

Preferred ranges of the mean molecular weight are, e.g., 4 to 70 kD or10 to 70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kDor 10 to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18kD or 12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD.

According to particularly preferred embodiments of the presentinvention, the mean molecular weight of hydroxyethyl starch employed isin the range of from more to than 4 kD and below 70 kD, such as about 10kD, or in the range of from 9 to 10 kD or from 10 to 11 kD or from 9 to11 kD, or about 12 kD, or in the range of from 11 to 12 kD or from 12 to13 kD or from 11 to 13 kD, or about 18 kD, or in the range of from 17 to18 kD or from 18 to 19 kD or from 17 to 19 kD, or about 50 kD, or in therange of from 49 to 50 kD or from 50 to 51 kD or from 49 to 51 kD.

As to the upper limit of the molar degree of substitution (DS), valuesof up to 2.0 such as 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9 or 2.0 are also possible, values of below 2.0 being preferred,values of below 1.5 being more preferred, values of below 1.0 such as0.7, 0.8 or 0.9 being still more preferred.

Therefore, preferred ranges of the molar degree of substitution are from0.1 to 2 or from 0.1 to 1.5 or from 0.1 to 1.0 or from 0.1 to 0.9 orfrom 0.1 to 0.8. More preferred ranges of the molar degree ofsubstitution are from 0.2 to 2 or from 0.2 to 1.5 or from 0.2 to 1.0 orfrom 0.2 to 0.9 or from 0.2 to 0.8. Still more preferred ranges of themolar degree of substitution are from 0.3 to 2 or from 0.3 to 1.5 orfrom 0.3 to 1.0 or from 0.3 to 0.9 or from 0.3 to 0.8. Even morepreferred ranges of the molar degree of substitution are from 0.4 to 2or from 0.4 to 1.5 or from 0.4 to 1.0 or from 0.4 to 0.9 or from 0.4 to0.8.

As far as the degree of substitution (DS) is concerned, DS is preferablyat least 0.1, more preferably at least 0.2, more preferably at least 0.3and more preferably at least 0.4. Preferred ranges of DS are from 0.1 to0.8, more preferably from 0.2 to 0.8, more preferably from 0.3 to 0.8and even more preferably from 0.4 to 0.8, still more preferably from 0.1to 0.7, more preferably from 0.2 to 0.7, more preferably from 0.3 to 0.7and more preferably from 0.4 to 0.7. Particularly preferred values of DSare, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, with 0.2, 0.3, 0.4,0.5, 0.6, 0.7 or 0.8 being more preferred, 0.3, 0.4, 0.5, 0.6, 0.7 or0.8 being even more preferred, 0.4, 0.5, 0.6, 0.7 or 0.8 being stillmore preferred and, e.g. 0.4 and 0.7 being particularly preferred.

In the context of the present invention, a given value of the molardegree of substitution such as 0.8 may be the exact value or may beunderstood as being in a range of from 0.75 to 0.84. Therefore, forexample, a given value of 0.1 may be the exact value of 0.1 or be in therange of from 0.05 to 0.14, a given value of 0.4 may be the exact valueof 0.4 or in the range of from 0.35 to 0.44, or a given value of 0.7 maybe the exact value of 0.7 or be in the range of from 0.65 to 0.74.

Particularly preferred combinations of molecular weight of thehydroxyalkyl starch, preferably hydroxyethyl starch, and its degree ofsubstitution DS are, e.g., 10 kD and 0.4 or 10 kD and 0.7 or 12 kD and0.4 or 12 kD and 0.7 or 18 kD and 0.4 or 18 kD and 0.7 or 50 kD and 0.4or 50 kD and 0.7 or 100 kD and 0.7.

An example of HES having a mean molecular weight of about 130 kD is aHES with a degree of substitution of 0.2 to 0.8 such as 0.2, 0.3, 0.4,0.5, 0.6, 0.7, or 0.8, preferably of 0.4 to 0.7 such as 0.4, 0.5, 0.6,or 0.7.

An example for HES with a mean molecular weight of about 130 kD isVoluven® from Fresenius. Voluven® is an artifical colloid, employed,e.g., for volume replacement used in the therapeutic indication fortherapy and prophylaxis of hypovolaemia. The characteristics of Voluven®are a mean molecular weight of 130,000+/−20,000 D, a molar substitutionof 0.4 and a C2:C6 ratio of about 9:1.

As far as the ratio of C₂:C₆ substitution is concerned, saidsubstitution is preferably in the range of from 2 to 20, more preferablyin the range of from 2 to 15 and even more preferably in the range offrom 3 to 12.

According to a further embodiment of the present invention, alsomixtures of hydroxyethyl starches may be employed having different meanmolecular weights and/or different degrees of substitution and/ordifferent ratios of C₂:C₆ substitution. Therefore, mixtures ofhydroxyethyl starches may be employed having different mean molecularweights and different degrees of substitution and different ratios ofC₂:C₆ substitution, or having different mean molecular weights anddifferent degrees of substitution and the same or about the same ratioof C₂:C₆ substitution, or having different mean molecular weights andthe same or about the same degree of substitution and different ratiosof C₂:C₆ substitution, or having the same or about the same meanmolecular weight and different degrees of substitution and differentratios of C₂:C₆ substitution, or having different mean molecular weightsand the same or about the same degree of substitution and the same orabout the same ratio of C₂:C₆ substitution, or having the same or aboutthe same mean molecular weights and different degrees of substitutionand the same or about the same ratio of C₂:C₆ substitution, or havingthe same or about the same mean molecular weight and the same or aboutthe same degree of substitution and different ratios of C₂:C₆substitution, or having about the same mean molecular weight and aboutthe same degree of substitution and about the same ratio of C₂:C₆substitution.

In different conjugates and/or different methods according to thepresent invention, different hydroxyalkyl starches, preferably differenthydroxyethyl starches and/or different hydroxyalkyl starch mixtures,preferably different hydroxyethyl starch mixtures, may be employed.

In the context of the present invention, the term “reactive carboxygroup” refers to a reactive ester, a carboxylic acid anhydride,isothiocyanates or isocyanate may be mentioned. Preferred reactiveesters are derived from N-hydroxy succinimides such as N-hydroxysuccinimide or Sulfo-N-hydroxy succinimide, suitably substituted phenolssuch as p-nitrophenol, o,p-dinitrophenol, o,o′-dinitrophenol,trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol,trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol,pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxybenzotriazole. Especially preferred are N-hydroxy succinimides, withN-hydroxy succinimide and Sulfo-N-hydroxy succinimide being especiallypreferred. All alcohols may be employed alone or as suitable combinationof two or more thereof. As reactive ester, pentafluorophenyl ester andN-hydroxy succinimide ester are especially preferred.

As to the group R′ of the thioester group —(C═Y)—S—R′, preferably—(C═O)—S—R′, no specific limitations exist given that the group —S—R′forms an electrophilic leaving group which is suitable for adisplacement when reacted with the alpha-X beta-amino group. Preferredresidues SR′ include, but are not limited to, substituents which arederived from substituted or unsubstituted thiophenol, thiopyridine,benzyl mercaptane, ethanethiol, methanethiol, 2-mercaptoethansulfonicacid, 2-mercaptoacetic acid, 2-mercaptoacetic acid methyl or ethylester, 3-mercaptopropionic acid, 3-mercaptopropionic acid methyl orethyl ester, 4-mercaptobutyric acid, 4-mercaptobutyric acid methyl orethyl ester.

According to a first alternative of the present invention, the thioestergroup is comprised in the hydroxyalkyl starch and the alpha-X beta-aminogroup is comprised in the active substance.

Therefore, the present invention also relates to a method as describedabove, wherein a thioester functionalized hydroxyalkyl starch is reactedwith an alpha-X beta-amino group of the active substance.

The thioester functionalized hydroxyalkyl starch, preferablyhydroxyethyl starch, can be provided by every suitable method.

According to one embodiment of the present invention, the methodaccording to the invention comprises selectively oxidizing hydroxyalkylstarch at its reducing end to give hydroxalkyl starch according toformula (IIIa)

and/or according to formula (IIIb)

and reacting hydroxyalkyl starch selectively oxidized at its reducingend with at least one suitable compound to give a thioesterfunctionalized hydroxyalkyl starch.

Oxidation of the hydroxyalkyl starch, preferably hydroxyethyl starch,may be carried out according to each method or combination of methodswhich result in compounds having the above-mentioned structures (IIIa)and/or (IIIb). Although the oxidation may be carried out according toall suitable method or methods resulting in the oxidized reducing end ofhydroxyalkyl starch, it is preferably carried out using an alkalineiodine solution as described, e.g., in DE 196 28 705 A1 the respectivecontents of which (example A, column 9, lines 6 to 24) is incorporatedherein by reference.

According to one alternative of the present invention, the hydroxyalkylstarch, preferably selectively oxidized at its reducing end, is reactedwith an at least bifunctional compound comprising a functional group Mwhich is reacted with the hydroxyalkyl starch, preferably at theoxidized reducing end, and a functional group Q which is a thioestergroup or a functional group which can be modified to give a thioestergroup.

As functional group M of the at least bifunctional compound which isreacted with the hydroxyalkyl starch, especially a group is to bementioned having the structure R′-NH— where R′ is hydrogen or a alkyl,cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylarylresidue where the cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl orcycloalkylaryl residue may be linked directly to the NH group or,according to another embodiment, may be linked by an oxygen bridge tothe NH group. The alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl,alkaryl, or cycloalkylaryl residues may be suitably substituted. Aspreferred substituents, halogens such as F, Cl or Br may be mentioned.Especially preferred residues R′ are hydrogen, alkyl and alkoxy groups,and even more preferred are hydrogen and unsubstituted alkyl and alkoxygroups.

Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 Catoms are preferred. More preferred are methyl, ethyl, propyl,isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especiallypreferred are methyl, ethyl, methoxy, ethoxy, and particular preferenceis given to methyl or methoxy.

According to another embodiment of the present invention, the functionalgroup M has the structure R′-NH—R″- where R″ preferably comprises thestructure unit —NH— and/or the structure unit —(C=G)- where G is O or S,and/or the structure unit —SO₂—. Specific examples for the functionalgroup R″ are

-   -   And

where, if G is present twice, it is independently O or S.

Therefore, the present invention also relates to a method and aconjugate as mentioned above wherein the functional group M is selectedfrom the group consisting of

wherein G is O or S and, if present twice, independently O or S, and R′is methyl.

According to a particularly preferred embodiment of the presentinvention, the functional group M is an amino group —NH₂.

According to the group Q which is a thioester group or a functionalgroup which 1o can be chemically modified to give a thioester group, thefollowing functional groups are to be mentioned, among others:

-   -   C—C-double bonds or C—C-triple bonds or aromatic C—C-bonds;    -   the thio group or the hydroxy groups;    -   alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;    -   1,2-dioles;        -   1,2 amino-thioalcohols;        -   azides;    -   1,2-aminoalcohols;    -   the amino group —NH₂ or derivatives of the amino groups        comprising the structure unit —NH— such as aminoalkyl groups,        aminoaryl group, aminoaralkyl groups, or alkarlyaminogroups;    -   the hydroxylamino group —O—NH₂, or derivatives of the        hydroxylamino group comprising the structure unit —O—NH—, such        as hydroxylalkylamino groups, hydroxylarylamino groups,        hydroxylaralkylamino groups, or hydroxalalkarylamino groups;    -   alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups,        or alkaryloxyamino groups, each comprising the structure unit        —NH—O—; residues having a carbonyl group, -Q-C(=G)-M, wherein G        is O or S, and M is, for example,    -   —OH or —SH;    -   an alkoxy group, an aryloxy group, an aralkyloxy group, or an        alkaryloxy group;    -   an alkylthio group, an arylthio group, an aralkylthio group, or        an alkarylthio group;    -   an alkylcarbonyloxy group, an arylcarbonyloxy group, an        aralkylcarbonyloxy group, an alkarylcarbonyloxy group;    -   activated esters such as esters of hydroxylamines having imid        structure such as N-hydroxysuccinimide or having a structure        unit O—N where N is part of a heteroaryl compound or, with G=O        and Q absent, such as aryloxy compounds with a substituted aryl        residue such as pentafluorophenyl, paranitrophenyl or        trichlorophenyl;

wherein Q is absent or NH or a heteroatom such as S or O;

-   -   —NH—NH₂, or —NH—NH—;    -   —NO₂;    -   the nitril group;    -   carbonyl groups such as the aldehyde group or the keto group;        -   the carboxy group;        -   the —N═O group or the —N═C═S group;    -   vinyl halide groups such as the vinyl iodide or the vinyl        bromide group or triflate;        -   —≡C—H;        -   —(C═NH₂Cl)—OAlkyl        -   groups —(C═O)—CH₂-Hal wherein Hal is Cl, Br, or I;        -   —CH═CH—SO₂—;        -   a disulfide group comprising the structure —S—S—;    -   the group

-   -   the group

According to a first alternative, the functional group Q is a thioestergroup.

According to a second alternative, the functional group Q is a groupwhich is further modified to give a thioester group. According to thisembodiment, the hydroxyalkyl starch derivative resulting from thereaction of hydroxyalkyl starch, preferably selectively oxidized at itsreducing end, with the at least bifunctional compound, is reacted with afurther at least bifunctional compound comprising a functional groupwhich is reacted with the functional group Q of the hydroxyalkyl starchderivative and a thioester group.

As functional group of the further compound which is reacted with thefunctional group Q, every suitable functional group from the list offunctional groups described above for functional Q is to be mentionedgiven that it can be reacted with Q.

According to one embodiment of the present invention, the functionalgroup Q comprises the chemical structure —NH—.

According to a preferred embodiment of the present invention, thefunctional group Q is a group having the structure R′-NH— where R′ ishydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkarylor cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directlyto the NH group or, according to another embodiment, may be linked by anoxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitablysubstituted. As preferred substituents, halogenes such as F, Cl or Brmay be mentioned. Especially preferred residues R′ are hydrogen, alkyland alkoxy groups, and even more preferred are hydrogen andunsubstituted alkyl and alkoxy groups.

Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 Catoms are preferred. More preferred are methyl, ethyl, propyl,isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especiallypreferred are methyl, ethyl, methoxy, ethoxy, and particular preferenceis given to methyl or methoxy.

According to another embodiment of the present invention, the functionalgroup

Q has the structure R′-NH—R″- where R″ preferably comprises thestructure unit —NH— and/or the structure unit —(C=G)- where G is O or S,and/or the structure unit —SO₂—. According to more preferredembodiments, the functional group R″ is selected from the groupconsisting of

-   -   And

where, if G is present twice, it is independently O or S.

Therefore, the present invention also relates to a method and aconjugate as mentioned above wherein the functional group Q is selectedfrom the group consisting of

wherein G is O or S and, if present twice, independently O or S, and R′is methyl.

According to a particularly preferred embodiment of the presentinvention, the functional group Q is an amino group —NH₂.

According to one embodiment of the present invention, both M and Qcomprise an amino group —NH—. According to a preferred embodiment, bothM and Q are an amino group —NH₂.

According to a preferred embodiment of the present invention, thecompound comprising M and Q is a homobifunctional compound, morepreferably a homobifunctional compound comprising, as functional groupsM and Q, most preferably the amino group —NH₂, or according to otherembodiments, the hydroxylamino group —O—NH₂ or the group

with G preferably being O. Specific examples for these compoundscomprising M and Q are

In the preferred case both M and Q are an amino group —NH₂, M and Q maybe separated by any suitable spacer. Among others, the spacer may be anoptionally substituted, linear, branched and/or cyclic hydrocarbonresidue. Generally, the hydrocarbon residue has from 1 to 60, preferablyfrom 1 to 40, more preferably from 1 to 20, more preferably from 2 to10, more preferably from 2 to 6 and especially preferably from 2 to 4carbon atoms. If heteroatoms are present, the separating group comprisesgenerally from 1 to 20, preferably from 1 to 8 and especially preferablyfrom 1 to 4 heteroatoms. The hydrocarbon residue may comprise anoptionally branched alkyl chain or an aryl group or a cycloalkyl grouphaving, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, analkaryl group where the alkyl part may be a linear and/or cyclic alkylgroup. According to an even more preferred embodiment, the hydrocarbonresidue is an alkyl chain of from 1 to 20, preferably, from 2 to 10,more preferably from 2 to 6, and especially preferably from 2 to 4carbon atoms.

Therefore, the present invention also relates to a method and aconjugate as described above, wherein the hydroxyalkyl starch is reactedwith 1,4-diaminobutane, 1,3-diaminopropane or 1,2-diaminoethane to givean amino functionalized hydroxyalkyl starch derivative.

According to an alternative of the present invention, the hydroxyalkylstarch is employed with oxidized reducing end. According to anotheralternative of the present invention, the hydroxyalkyl starch isemployed with non-oxidized reducing end. According to a furtheralternative of the present invention, the hydroxyalkyl starch is reactedwith functional group M via the selectively oxidized reducing end.According to a further alternative of the present invention, thehydroxyalkyl starch is reacted with functional group M via thenon-oxidized reducing end. According to a further alternative of thepresent invention, the hydroxyalkyl starch is reacted with functionalgroup M at a suitable chemical moiety of the HAS molecule. According toa further alternative, hydroxyalkyl starch may be reacted withfunctional group M at the non-oxidized reducing end and at least onefurther chemical moiety of the HAS molecule or at the selectivelyoxidized reducing end and at least one further chemical moiety of theHAS molecule.

The term “the hydroxyalkyl starch is reacted via the reducing end” or“the hydroxyalkyl starch is reacted via the selectively oxidizedreducing end” as used in the context of the present invention relates toa process according to which the hydroxyalkyl starch is reactedpredominantly via its (selectively oxidized) reducing end.

This term “predominantly via its (selectively oxidized) reducing end”relates to processes according to which statistically more than 50%,preferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and still more preferably at least 95% such as95%, 96%, 97%, 98%, or 99% of the hydroxyalkyl starch molecules employedfor a given reaction are reacted via at least one (selectively oxidized)reducing end per hydroxyalkyl starch molecule, wherein a givenhydroxyalkyl starch molecule which is reacted via at least one reducingend can be reacted in the same given reaction via at least one furthersuitable functional group which is comprised in said hydroxyalkyl starchmolecule and which is not a reducing end. If one or more hydroxyalkylstarch molecule(s) is (are) reacted via at least one reducing andsimultaneously via at least one further suitable functional group whichis comprised in this (these) hydroxyalkyl starch molecule(s) and whichis not a reducing end, statistically preferably more than 50%,preferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and still more preferably at least 95% such as95%, 96%, 97%, 98%, or 99% of all reacted functional groups of thesehydroxyalkyl starch molecules, said functional groups including thereducing ends, are reducing ends.

The term “reducing end” as used in the context of the present inventionrelates to the terminal aldehyde group of a hydroxyalkyl starch moleculewhich may be present as aldehyde group and/or as corresponding acetalfrom. In case the reducing end is oxidized, the aldehyde or acetal groupis in the form of a carboxy group and/or of the corresponding lactone.

As described above, the amino functionalized hydroxyalkyl starchderivative is preferably further reacted with an at least bifunctionalcompound comprising a functional group which is reacted with the aminogroup of the amino functionalized hydroxyalkyl starch derivative, and athioester group.

As preferred compounds to be reacted with the amino functionalizedhydroxyalkyl starch derivative, compounds are to be mentioned whichcomprise a reactive carboxy group which is reacted with the amino group,and a thioester group. As reactive carboxy group, a reactive ester,isothiocyanates or isocyanate may be mentioned. Preferred reactiveesters are derived from N-hydroxy succinimides such as N-hydroxysuccinimide or Sulfo-N-hydroxy succinimide, suitably substituted phenolssuch as p-nitrophenol, o,p-dinitrophenol, o,o′-dinitrophenol,trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol,trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol,pentachlorophenol, pentafluorophenol, or hydroxyazoles such as hydroxybenzotriazole.

The reactive carboxy group and the thioester may be separated by anysuitable spacer. Among others, the spacer may be an optionallysubstituted, linear, branched and/or cyclic hydrocarbon residue.Generally, the hydrocarbon residue has from 1 to 60, preferably from 1to 40, more preferably from 1 to 20, more preferably from 2 to 10, morepreferably from 2 to 6 and especially preferably from 2 to 4 carbonatoms. If heteroatoms are present, the separating group comprisesgenerally from 1 to 20, preferably from 1 to 8 and especially preferablyfrom 1 to 4 heteroatoms. The hydrocarbon residue may comprise anoptionally branched alkyl chain or an aryl group or a cycloalkyl grouphaving, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, analkaryl group where the alkyl part may be a linear and/or cyclic alkylgroup. According to one embodiment of the present invention, thereactive carboxy group and the thioester group are separated by an alkylresidue with 2, 3, or 4 carbon atoms such as an ethylen group, propylenegroup, or butylene group.

As an example, in case hydroxyalkyl starch, preferably hydroxyethylstarch, is reacted with its oxidized reducing end with a diaminoalkanesuch as 1,4-diaminobutane, 1,3-diaminopropane, or 1,2-diaminoethane, andthe resulting hydroxyalkyl starch derivative is further reacted with acompound comprising a thioester group —S—R′ and a reactive carboxy groupwhere the thioester group and the reactive carboxy group are separatedby a ethylen group, propylene group, or butylene group, a thioesterfunctionalized hydroxyalkyl starch according to

may result, where n, m are independently 2, 3, 4.

Therefore, the present invention also relates to method and a conjugateas described above, said method comprising oxidizing hydroxyalkyl starchat its reducing end, reacting the oxidized reducing end with an at leastbifunctional compound comprising two amino groups to give an aminofunctionalized hydroxyalkyl starch derivative, and reacting the aminogroup of the derivative with an at least bifunctional compoundcomprising at least one functional group which is reacted with the aminogroup of the derivative, preferably a reactive carboxy group, andcomprising at least one thioester group, preferably one thioester group—SR′.

According to another embodiment of the present invention, a thioesterfunctionalized hydroxyalkyl starch is prepared by selectively oxidizinghydroxyalkyl starch at its reducing end, as described above, and

-   -   by converting the oxidized reducing end of the hydroxyalkyl        starch to an imidazolide of a carboxylic acid, prepared, e.g.,        by reaction of the corresponding carboxylic acid with, e.g.,        N,N-carbonyldiimidazole) with a comparatively acidic thiol        (Masamune, S., et al., J. Am. Chem. Soc. 98 (1976) 7874), or    -   by converting the oxidized reducing end of the hydroxyalkyl        starch using a disulfide and triphenylphosphine to the        corresponding thioester (Mukaiyama, T., et al., Bull. Chem. Soc.        Jpn. 43 (1970) 1271), or    -   reacting the oxidized reducing end of the hydroxyalkyl starch        with aryl thioisocyanates (Grieco, P., et al., Tetrahedron Lett.        43 (1979) 1283), or    -   reacting the oxidized reducing end of the hydroxyalkyl starch        with thiopyridyl chloroformate (Corey, E. J., et al.,        Tetrahedron Lett. (1979), 2875), or    -   reacting the oxidized reducing end of the hydroxyalkyl starch        with 2-fluoro-N-methylpyridinium tosylate and thiols (Watanabe,        Y., et al., Chem. Lett. (1976) 741), or    -   reacting the oxidized reducing end of the hydroxyalkyl starch        with a carbodiimide such as such as diisopropyl carbodiimde        (DIC), dicyclohexyl carbodimides (DCC),        1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), on a solid        support immobilized EDC and a thiol. (C. E-Lin et al.        Tetrahedron Lett. 43 (2002) 4531-34; M. Adamczyk, Tetrahedron        Lett. 37 (1996) 4305-8, J. Hovinen, Nucleosides Nucleotides        18 (1999) 1263-4).

Therefore, the present invention also relates to a method and aconjugate as described above, said method comprising oxidizinghydroxyalkyl starch at its reducing end and reacting the oxidizedreducing end with carbodiimide such as such as diisopropyl carbodiimde(DIC), dicyclohexyl carbodiimides (DCC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) being preferred anda thiol to give the thioester functionalized hydroxyalkyl starch.

The thioester functionalized hydroxyalkyl starch derivative as describedabove is then reacted with the alpha-X beta-amino group of the activesubstance.

Therefore, the present invention also relates to a conjugate asdescribed above according to formula (IV)

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the thioester group, and wherein AS′ is theresidue of the active substance or a derivative thereof which was linkedto the alpha-X beta-amino group.

According to a preferred embodiment of the present invention, thealpha-X beta-amino group is comprised in a cysteine residue or ahistidine residue of the active substance, preferably of the smallmolecule drug, peptide, or protein.

Therefore, the present invention also relates to a method and aconjugate as described above, wherein the alpha-X beta-amino group iscomprised in a cysteine residue or a histidine residue of the activesubstance.

More preferably, the cysteine or histidine residue of the activesubstance is an N-terminal cysteine or histidine of a peptide or aproteine. The N-terminal cysteine or histidine residue can be anaturally occuring N-terminal cysteine or histidine residue or can beN-terminally introduced in the peptide or protein by a suitablemodification of the peptide or protein sequence. In peptides, thementioned amino acids can be introduced during synthesis. Peptidessynthesis is know in the art. (W. Chang, P. D. White ; Fmoc solid phasepeptide synthesis, a practical approach; Oxford University Press,Oxford, 2000, ISBN 0199637245).

Recombinant polypeptides are obtainable by way of standard molecularbiological techniques as, for example, described in Molecular Cloning: ALaboratory Manual, 3rd edition, Eds. Sambrook et al., CSHL Press 2001.Briefly, polypeptides can be expressed from recombinant expressionvectors comprising a nucleic acid encoding the desired polypeptide,which nucleic acid is operably linked to at least one regulator sequenceallowing expression of the desired polypeptide. For example, a nucleicacid sequence encoding the desired polypeptide can be isolated andcloned into an expression vector and the vector can then be transformedinto a suitable host cell for expression of the desired polypeptide.Such a vector can be a plasmid, a phagemid or a cosmid. For example, anucleic acid molecule can be cloned in a suitable fashion intoprokaryotic or eukaryotic expression vectors (Molecular Cloning, seeabove). Such expression vectors comprise at least one promoter and canalso comprise a signal for translation initiation and—in the case ofprokaryotic expression vectors—a signal for translation termination,while in the case of eukaryotic expression vectors preferably expressionsignals for transcriptional termination and for polyadenylation arecomprised. Examples for prokaryotic expression vectors are, forexpression in Escherichia coli e.g. expression vectors based onpromoters recognized by T7 RNA polymerase as described in U.S. Pat. No.4,952,496, for eukaryotic expression vectors for expression inSaccharomyces cerevisiae, e.g. the vectors G426/Met25 or P526/Gal1(Mumberg et al., (1994) Nucl. Acids Res., 22, 5767-5768), for theexpression in insect cells, e.g. Baculovirus vectors, as e.g describedin EP-B1-0127839 or EP-B1-0549721 or by Ciccarone et al. (“Generation ofrecombinant Baculovirus DNA in E. coli using baculovirus shuttle vector”(1997) Volume 13, U. Reischt, ed. (Totowa, N.J.: Humana Press Inc.) andfor the expression in mammalian cells, e.g. the vectors Rc/CMW andRc/ASW and SW40-Vectors, which are commonly known and commerciallyavailable, or the EBNA-system described in Example 4, the Sindbisreplicon-based pCytTS (Boorsma et al. (2002) Biotechnol. Bioeng. 79(6):602-609), the Sindbis virus expression system (Schlesinger (1993) TrendsBiotechnol. 11(1):18-22) or an Adenovirus expression system (He et al.(1998) Proc. Natl. Acad. Sci. USA 95:2509-2514). The molecularbiological methods for the production of these expression vectors aswell as the methods for transfecting host cells and culturing suchtransfected host cells as well as the conditions for producing andobtaining the polypeptides of the invention from said transformed hostcells are well known to the skilled person.

Polypeptides with the desired N-terminal Cys or His residue can begenerated from the polypeptides expressed and purified as describedabove by cloning the polypeptide of interest behind an N-terminal leadersequence which is removable to yield the polypeptides with the desiredN-terminal Cys or His residue.

This can be achieved, for example, by proteolytic cleavage of thepolypeptide expressed and purified as described above. In such a case, afusion polypeptide was cloned, expressed and purified wherein a Cys orHis residue follows directly a highly selective protease cleavage site,for example a His or Cys residue immediately following the Factor Xacleavage site: Ile (Glu/Asp) Gly Arg|(Cys/His), or a His or Cys residueimmediately following the Enterokinase cleavage site: Asp Asp Asp AspLys|(Cys/His), wherein | denotes the site of cleavage by the protease.

This can further be achieved, for example, by cleavage of thepolypeptide during expression, for example cleavage at the stage of ERtranslocation by the signal peptidase. In such a case, a fusionpolypeptide was cloned and expressed wherein a Cys or His residuefollows directly the signal peptide directing the recombinantpolypeptide to the secretory pathway (for review see Rapoport et al.,Annu Rev Biochem. 1996; 65:271-303).

The molecular biological methods to manipulate the coding sequence of arecombinant polypeptide so that a coding sequence for a polypeptide withthe desired Cys or His residue at the desired position is generated arewell known in the art (Sambrook, above).

According to the native chemical ligation reaction according to thepresent invention, the intermediate product of the reaction of thethioester functionalized hydroxyalkyl starch and the alpha-X beta-aminofunctionalized active substance, preferably a peptide or protein with acysteine or histidine residue, preferably a N-terminal cysteine orhistidine residue, is a thioester which is irreversibly converted byintramolecular trans-acetylation to the inventive conjugate comprisingthe hydroxyalkyl starch derivative linked via an amide linkage to theactive substance.

According to another alternative, the present invention also relates toa method as described above, wherein a thioester functionalized activesubstance is reacted with an alpha-X beta-amino group of a hydroxyalkylstarch derivative.

Accordingly, the present invention also relates to a conjugate asdescribed above, having a structure according to formula (V)

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the alpha-X beta-amino group, and whereinAS′ is the residue of the active substance or a derivative thereof whichwas linked to the thioester group.

The alpha-X beta-amino functionalized hydroxyalkyl starch can beprepared by any suitable method. According to one embodiment of thepresent invention, the alpha-X beta-amino functionalized hydroxyalkylstarch is prepared by a method comprising selectively oxidizing thehydroxyalkyl starch at its reducing end and suitably chemicallymodifying the oxidized reducing end to give an alpha-X beta-aminofunctionalized hydroxyalkyl starch derivative.

Therefore, according to one embodiment of the present invention, themethod according to the invention comprises selectively oxidizinghydroxyalkyl starch at its reducing end to give hydroxalkyl starchaccording to formula to formula (IIIa)

and/or according to formula (IIIb)

and suitably reacting hydroxyalkyl starch selectively oxidized at itsreducing end to give a thioester functionalized hydroxyalkyl starch.

Oxidation of the hydroxyalkyl starch, preferably hydroxyethyl starch,may be carried out according to each method or combination of methodswhich result in compounds having the above-mentioned structures (IIIa)and/or (IIIb). Although the oxidation may be carried out according toall suitable method or methods resulting in the oxidized reducing end ofhydroxyalkyl starch, it is preferably carried out using an alkalineiodine solution as described, e.g., in DE 196 28 705 A1 the respectivecontents of which (example A, column 9, lines 6 to 24) is incorporatedherein by reference.

According to one embodiment of the present invention, the hydroxyalkylstarch selectively oxidized at its reducing end is reacted with an atleast bifunctional compound comprising a functional group Z which isreacted with the oxidized reducing end and a functional group W which isfurther reacted with a compound which comprises a functional group Vbeing reacted with W and which comprises an alpha-X beta-amino group.

Therefore, the present invention also relates to a method as describedabove, said method comprising oxidizing hydroxyalkyl starch at itsreducing end, reacting the oxidized reducing end with a functional groupZ of a compound comprising, in addition to Z, a further functional groupW, to give a first hydroxyalkyl starch derivative, and reacting thefunctional group W of the first hydroxyalkyl starch derivative with afunctional group V of a compound comprising, in addition to V, analpha-X beta-amino group, to give the alpha-X beta-amino functionalizedhydroxyalkyl starch derivative.

According to one embodiment, both Z and W comprise the chemicalstructure —NH—.

According to a preferred embodiment of the present invention, thefunctional groups Z and W are a group having the structure R′-NH— whereR′ is hydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl,alkaryl or cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directlyto the NH group or, according to another embodiment, may be linked by anoxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitablysubstituted. As preferred substituents, halogenes such as F, Cl or Brmay be mentioned. Especially preferred residues R′ are hydrogen, alkyland alkoxy groups, and even more preferred are hydrogen andunsubstituted alkyl and alkoxy groups.

Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 Catoms are preferred. More preferred are methyl, ethyl, propyl,isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especiallypreferred are methyl, ethyl, methoxy, ethoxy, and particular preferenceis given to methyl or methoxy.

According to another embodiment of the present invention, the functionalgroups Z and W have the structure R′-NH—R″- where R″ preferablycomprises the structure unit —NH— and/or the structure unit —(C=G)-where G is O or S, and/or the structure unit —SO₂—. According to morepreferred embodiments, the functional group R″ is selected from thegroup consisting of

-   -   and

where, if G is present twice, it is independently O or S.

Therefore, the present invention also relates to a method and aconjugate as mentioned above wherein the functional groups Z and W areindependently selected from the group consisting of

wherein G is O or S and, if present twice, independently O or S, and R′is methyl.

Specific examples for these compounds comprising Z and W are

According to a particularly preferred embodiment of the presentinvention, both functional groups Z and W are an amino group —NH₂.

Therefore, the present invention also relates to a method as describedabove, wherein the compound comprising Z and W is a diaminofunctionalized compound.

In case both Z and W are an amino group —NH₂, Z and W may be separatedby any suitable spacer. Among others, the spacer may be an optionallysubstituted, linear, branched and/or cyclic hydrocarbon residue.Generally, the hydrocarbon residue has from 1 to 60, preferably from 1to 40, more preferably from 1 to 20, more preferably from 2 to 10, morepreferably from 2 to 6 and especially preferably from 2 to 4 carbonatoms. If heteroatoms are present, the separating group comprisesgenerally from 1 to 20, preferably from 1 to 8 and especially preferablyfrom 1 to 4 heteroatoms. The hydrocarbon residue may comprise anoptionally branched alkyl chain or an aryl group or a cycloalkyl grouphaving, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, analkaryl group where the alkyl part may be a linear and/or cyclic alkylgroup. According to an even more preferred embodiment, the hydrocarbonresidue is an alkyl chain of from 1 to 20, preferably from 2 to 10, morepreferably from 2 to 6, and especially preferably from 2 to 4 carbonatoms.

Therefore, the present invention also relates to a method and aconjugate as described above, wherein the hydroxyalkyl starch,preferably at its oxidized reducing end, is reacted with1,4-diaminobutane, 1,3-diaminopropane or 1,2-diaminoethane to give anamino functionalized hydroxyalkyl starch derivative.

Thus, according to a first alternative, the functional group Z being anamino group NH₂ is reacted with the oxidized reducing end of thehydroxyalkyl starch resulting in an amido group linking the hydroxyalkylstarch and the compound comprising Z and W.

According to a second alternative, the functional group Z being an aminogroup NH₂ is reacted with the non-oxidized reducing end of thehydroxyalkyl starch via reductive amination resulting in an imino groupwhich is subsequently preferably hydrogenated to give a amino group, theimino group and the amino group, respectively, linking the hydroxyalkylstarch and the compound comprising Z and the amino group W. In this caseit is preferred that the compound comprising Z and W is a primary aminewhich contains—as functional group—only one amino group. In thisspecific case, although the compound contains only one functional group,it is regarded as bifunctional compound comprising Z and W wherein Z isthe amino group contained in the compound subjected to the reductiveamination with the reducing end of the polymer, and wherein W is thesecondary amino group resulting from the reductive amination andsubsequent hydrogenation.

According to a third alternative, the non-oxidized reducing end of thepolymer is reacted with ammonia via reductive amination resulting in aterminal imino group of the polymer which is subsequently preferablyhydrogenated to give a terminal amino group of the polymer and thus aterminal primary amino group. In this specific case, ammonia is regardedas bifunctional compound comprising Z and W wherein Z is NH₂ comprisedin the ammonia employed, and wherein W is the primary amino groupresulting from reductive amination and subsequent hydrogenation.

According to the present invention, the preferred amino functionalizedhydroxyalkyl starch derivative is further reacted with a compound whichcomprises a functional group V being reacted with W and which comprisesan alpha-X beta-amino group.

According to a preferred embodiment, the compound comprising afunctional group V being reacted with W and an alpha-X beta-amino groupis a cysteine or histidine derivative.

According to a further preferred embodiment, the cysteine derivative orhistidine derivative is pre-activated by a suitable activating agent.Suitable activating agents are, among others, carbodiimides such asdiisopropyl carbodiimde (DIC), dicyclohexyl carbodiimides (DCC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), with diisopropylcarbodiimde (DIC) being especially preferred.

The SH group and the amino group of the cysteine derivate should besuitably protected when the cysteine derivative is reacted with theamino group of the hydroxyalkyl starch derivative. As to the respectiveprotective groups, all suitable compounds known in the art may beemployed. Accordingly, if necessary, the group

should be suitably protected.

As protective group for the SH group, Acetamidomethyl-(Acm),tert-Butylthio-(StBu), 4-Methoxybenzyl-(4-MeOBzl),4-Methoxyltrityl-(Mmt), Trityl (Trt)- and 4-Methyltrityl-(Mtt) arepreferred, StBu being especially preferred.

As protective group for the amino group, tert-Butyloxycarbonyl-(Boc),Fluorenyloxycarbonyl-(Fmoc), 4-Methoxyltrityl-(Mmt), Trityl (Trt)- and4-Methyltrityl-(Mtt) are preferred Fmoc being especially preferred.

As protective group for group, tert-Butyloxycarbonyl-(Boc),Benzyloxymethyl (Born), Dinitrophenyl-(Dnp), 4-Methyltrityl-(Mtt),Tosyl-(Tos) and Trityl (Trt)- are preferred, 4-Methyltrityl-(Mtt) andTrityl (Trt) being especially preferred.

Therefore, the present invention also relates to a method as describedabove, wherein the compound comprising V and the alpha-X beta-amino iscysteine or histidine or a derivative thereof, V being the carboxygroup.

Therefore, the present invention also relates to an alpha-X beta-aminofunctionalized hydroxyalkyl starch derivative according to formula (VIc)

wherein L is an optionally suitably substituted, linear, cyclic and/orbranched alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl residue with2 to 10 carbon atoms in the respective alkyl residue,

wherein HAS″ refers to the HAS molecule without the terminal saccharideunit at the reducing end, and

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, preferably a hydroxyethyl group.

According to another embodiment, the present invention also relates to amethod as described above, wherein hydroxyalkyl starch is selectivelyoxidized at its reducing end, preferably according the method asdescribed above, and the resulting hydroxyalkyl starch is reacted with afunctional group Z of a compound comprising, in addition to Z, analpha-X beta-amino group.

Therefore, the present invention also relates to a method as describedabove, said method comprising oxidizing hydroxyalkyl starch at itsreducing end and reacting the oxidized reducing end with a functionalgroup Z of a compound comprising, in addition to Z, an alpha-Xbeta-amino group.

According to a preferred embodiment, Z comprises the chemical structure—NH—.

According to a preferred embodiment of the present invention, thefunctional group Z is a group having the structure R′-NH— where R′ ishydrogen or a alkyl, cycloalkyl, aryl, aralkyl, arylcycloallcyl, alkarylor cycloalkylaryl residue where the cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl or cycloalkylaryl residue may be linked directlyto the NH group or, according to another embodiment, may be linked by anoxygen bridge to the NH group. The alkyl, cycloalkyl, aryl, aralkyl,arylcycloalkyl, alkaryl, or cycloalkylaryl residues may be suitablysubstituted. As preferred substituents, halogenes such as F, Cl or Brmay be mentioned. Especially preferred residues R′ are hydrogen, alkyland alkoxy groups, and even more preferred are hydrogen andunsubstituted alkyl and alkoxy groups.

Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 Catoms are preferred. More preferred are methyl, ethyl, propyl,isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especiallypreferred are methyl, ethyl, methoxy, ethoxy, and particular preferenceis given to methyl or methoxy.

According to another embodiment of the present invention, the functionalgroup Z has the structure R′-NH—R″- where R″ preferably comprises thestructure unit —NH— and/or the structure unit —(C=G)- where G is O or S,and/or the structure unit —SO₂—. According to more preferredembodiments, the functional group R″ is selected from the groupconsisting of

-   -   and

where, if G is present twice, it is independently O or S.

According to a particularly preferred embodiment of the presentinvention, the functional group Z is an amino group —NH₂.

The amino group Z and the alpha-thiol beta-amino group may be separatedby any suitable spacer. Among others, the spacer may be an optionallysubstituted, linear, branched and/or cyclic hydrocarbon residue.Generally, the hydrocarbon residue has from 1 to 60, preferably from 1to 40, more preferably from 1 to 20, more preferably from 1 to 10, morepreferably from 1 to 6 and especially preferably from 1 to 4 carbonatoms. If heteroatoms are present, the separating group comprisesgenerally from 1 to 20, preferably from 1 to 8 and especially preferablyfrom 1 to 4 heteroatoms. The hydrocarbon residue may comprise anoptionally branched alkyl chain or an aryl group or a cycloalkyl grouphaving, e.g., from 5 to 7 carbon atoms, or be an aralkyl group, analkaryl group where the alkyl part may be a linear and/or cyclic alkylgroup. According to an even more preferred embodiment, the hydrocarbonresidue is an alkyl chain of from 1 to 4 such as a methylene, ethylene,propylene, or butylene residue. Particularly preferred is the methyleneresidue.

Therefore, the present invention also relates to a method as describedabove, wherein the compound comprising Z and the alpha-X beta-aminogroup is 1,3-diamino-2-thiol propane or 2,3-diamino-1-thiol propane,wherein the thiol group may be replaced by the group

Accordingly, the present invention also relates to an alpha-thiolbeta-amino functionalized hydroxyalkyl starch derivative according toformula (VIa)

or according to formula (VIb)

wherein HAS″ refers to the HAS molecule without the terminal saccharideunit at the reducing end, and

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, preferably a hydroxyethyl group.

The alpha-X beta-amino functionalized hydroxyalkyl starch derivative isthen reacted with the thioester functionalized active substance.

According to the native chemical ligation reaction according to thepresent invention, the intermediate product of the reaction of thethioester functionalized active substance and the alpha-X beta-aminofunctionalized hydroxyalkyl starch, is a thioester which is irreversiblyconverted by intramolecular trans-acetylation to the inventive conjugatecomprising the active substance linked via an amide linkage to thehydroxyalkyl starch derivative.

The reaction of the thioester functionalized compound, i.e. thethioester functionalized active substance or the thioesterfunctionalized hydroxyalkyl starch or derivative thereof, with thealpha-X beta-amino functionalized compound. i.e. the alpha-X beta-aminofunctionalized hydroxyalkyl starch or derivative thereof or the alpha-Xbeta-amino functionalized active substance, may be carried out in anysuitable solvent or mixture of at least two solvents and at a suitablepH and at a suitable reaction temperature.

The reaction temperature is preferably in the range of from 0 to 40° C.,more preferably or from 10 to 30° C. such as about 20 to 25° C., like20° C., 21° C., 22°C., 23° C., 24° C., or 25° C.

The pH the reaction is carried out at is preferably in the range of from5 to 9, preferably of from 6 to 8 and more preferably of from 6.5 to7.5.

As buffer media, acetate buffers such as sodium acetate buffer, e.g.having a concentration of about 0.1 M, bicarbonate buffers such asammonium bicarbonate buffer, e.g. having a concentration of about 0.5 M,and/or phosphate buffers such as sodium phosphate buffer, e.g. having aconcentration of about 0.1 M may be mentioned.

The reaction time is preferably in the range of up to 48 h, morepreferably of from 1 to 48 h, more preferably of from 8 to 32 h and morepreferably of from 20 to 24 h.

The reaction may be carried out in any suitable solvent. A preferredsolvent is water. To the solution of the thioester functionalizedcompound and/or the alpha-X beta-amino functionalized compound, at leastone suitbable catalyst and/or at least one adjuvant may be added.

Preferred catalysts are, among others, thiophenol in a concentration ofabout, e.g., from 2 to 6% v/v, preferably of from 3 to 5% v/v, or benzylmercaptan, or MESNA in a concentration of about, e.g., from 0.4 to 0.6 Msuch as about 0.5 M, and/or tris(carboxyethyl)phosphine or a mixture oftwo or more thereof.

Preferred adjuvants are, among others, urea in a concentration of about1 to 8 M, preferably of from 1 to 7 M, more preferably of from 1 to 6 M,more preferably of from 1 to 5 M, more preferably of from 1 to 4 M, morepreferably of from 1 to 3 M such as about 2 M, or guanidiniumhydrochloride in a concentration of about 4 to 8 M, preferably of from 5to 7 M such as about 6 M, a suitable organic solvent such as DMF oracetonitrile in a concentration of about 20 to 40% v/v, preferably about25 to 35% v/v, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in a concentration of about from 2 to 8% v/v,preferably of from 3 to 7% v/v, more preferably of from 4 to 6% v/v suchas about 5% v/v, or sodium chloride in a concentration of about from 0.1to 0.5 M, preferably of from 0.2 to 0.4 M such as about 0.3 M.

The concentration of the thioester functionalized compound and thealpha-X beta-amino functionalized compound, respectively, in thereaction solution is preferably in the range of from 0.001 to 0.5 M,more preferably of from 0.02 to 0.4 M, more preferably of from 0.05 M to0.3 M, more preferably of from 0.1 to 0.2 M.

According to one embodiment of the present invention, the activesubstance is a small molecule drug comprising a carboxy group. In thiscase, the carboxy group of the active substance is suitably converted ina thioester group —S—R′, e.g. by

-   -   suitably converting the oxidized reducing end of the        hydroxyalkyl starch to an acid chloride and reacting the acid        chloride with a mercaptane R′-SH via alkylthio-de-halogenation        (J. March, Advanced Organic Chemistry, 4th edition, John Wiley        and Sons, New York (1992) 409, or other methods cited therein        (paragraph 0-37), or    -   converting the oxidized reducing end of the hydroxyalkyl starch        to an acid chloride by reaction with a thallium(I) salt of a        thiolate (Spessard, G., et al., Organic Synthesis Collection,        vol. 7, 87), or    -   by reacting the carboxylic acid-terminated hydroxyalkyl starch        by reaction of the acid with a dialkyl or diphenyl        phosphorochloridate to form an anhydride which can then be        converted to the corresponding thioester (Masamune, S., et al.,        Can. J. Chem. 53 (1975) 3693), or    -   by converting the oxidized reducing end of the hydroxyalkyl        starch to an imidazolide of a carboxylic acid, prepared, e.g.,        by reaction of the corresponding carboxylic acid with, e.g.,        N,N-carbonyldiimidazole) with a comparatively acidic thiol        (Masamune, S., et al., J. Am. Chem. Soc. 98 (1976) 7874), or    -   by converting the oxidized reducing end of the hydroxyalkyl        starch using a disulfide and triphenylphosphine to the        corresponding thioester (Mukaiyama, T., et al., Bull. Chem. Soc.        Jpn. 43 (1970) 1271), or    -   reacting the oxidized reducing end of the hydroxyalkyl with aryl        thioisocyanates (Grieco, P., et al., Tetrahedron Lett. 43 (1979)        1283), or    -   reacting the oxidized reducing end of the hydroxyalkyl with        thiopyridyl chloroformate (Corey, E. J., et al., Tetrahedron        Lett. (1979), 2875), or    -   reacting the oxidized reducing end of the hydroxyalkyl with        2-fluoro-N-methylpyridinium tosylate (Watanabe, Y., et al.,        Chem. Lett. (1976) 741), or    -   reacting the oxidized reducing end of the hydroxyalkyl with        1-hydroxybenzotriazole (Pelter, A., et al., J. Chem Soc., Perkin        trans I (1977) 1672).

It is preferred that the carboxy group of the active substance beconverted to a thioester group —S—R′ by

-   -   suitably converting the oxidized reducing end of the        hydroxyalkyl starch to an acid chloride and reacting the acid        chloride with a mercaptane R′-SH via alkylthio-de-halogenation        (J. March, Advanced Organic Chemistry, 4th edition, John Wiley        and Sons, New York (1992) 409, or other methods cited therein        (paragraph 0-37), or by    -   reacting the oxidized reducing end of the hydroxyalkyl starch        with a carbodiimide such as such as diisopropyl carbodiimde        (DIC), dicyclohexyl carbodiimides (DCC),        1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), on a solid        support immobilized EDC and a thiol. (C. E-Lin et al.        Tetrahedron Lett. 43 (2002) 4531-34; M. Adamczyk, Tetrahedron        Lett. 37 (1996) 4305-8, J. Hovinen, Nucleosides Nucleotides        18 (1999) 1263-4)

According to another embodiment of the present invention, the activesubstance is an artificially produced peptide. According to onepreferred alternative of the present invention, the thioesterfunctionalized peptide is prepared using a suitable synthesis resinallowing for thioester functionalized peptides.

Thioester functionalized peptides can be synthesized using Boc chemistryas well as Fmoc chemistry. Tam et al. describe synthesis of thioesterfunctionalized peptides using Boc chemistry on the acid-labileTrt-mercapto-propionyl-MBHA resin (Tam., J. P., et al., Proc. Natl.Acad. Sci. USA 92 (1995) 12485). According to an especially preferredembodiment, the Trt group is removed with TFA/DCM. Following washing andneutralization with DIPEA, the resin can be loaded with the C-terminalresidue of the peptide using DIPCDI/HOBt activation. It is preferredthat unreacted thiol sites are then capped with AcCl/DIPEA in DCM. Afterchain extension using standard Boc protocols, HF cleavage provides theside chain deprotected thioester functionalized peptide.

Li et al. describe the synthesis of thioester functionalized peptidesusing Fmoc chemistry (Li, X. et al., (1998) Tetrahedron Letters39(47):8669-8672). In a preferred embodiment, a mixture of1-methylpyrrolidine, hexamethyleneimine and HOBt is used as thedeblocking agent for removal of Fmoc, preferably in a mixture of NMP andDMSO, more preferably wherein the molar ratio of NMP and DMSO is from1:10 to 10:1, even more preferably from 1:5 to 5:1, most preferably from1:2 to 2:1. The resins to be used can be, for example, the resins citedin Li et al. or cited in Goldstein and Gelb (2000) Tetrahedron Letters41(16):2797-2800.

Another possibility for producing thioester functionalized peptides isalso based on Fmoc chemistry and makes use of a sulfamylbutyrylsafety-catch linker employing thiolytic cleavage, for example from the4-Sulfamylbutyryl NovaSyn TG resin (Shin, Y., et al., J. Am. Chem. Soc.121 (1999), 11684; Ingenito, R., et al., J. Am. Chem Soc. 121 (1999)11369). Synthesis resins modified with the mentioned linkers arecommercially available for both, the Boc chemistry and the Fmocchemistry (e.g. from Novabiochem, Merck Biosciences GmbH, Schwalbach,Germany).

Therefore, the present invention also relates to a method for producinga thioester functionalized peptide, said method comprising producing thepeptide by chemical solid phase peptide synthesis on a synthesis resinallowing for the C-terminal thioester formation in the final cleavingstep form the solid support after side chain deprotection.

According to a further embodiment of the present invention, the activesubstance is a protein.

The protein can be produced by chemical synthetic procedures or can beof any human or another mammalian source and can be obtained bypurification from naturally occurring sources like human or animal. Inthis case, it is possible the produce a derivative of the protein byreacting at least one functional group of the protein with a suitablecompound which comprises at least one functional group which is reactedwith a functional group of the protein and at least one other functionalgroup which is a thioester group or a group G which can be chemicallymodified to give a thioester group. Such a chemical modification may bea reaction of this functional group G with a further compound whichcomprises a functional group which is reacted with G and anotherfunctional group which is a thioester group —S—R′.

As functional group of the protein, a hydroxyl group, an amino group, athiol group, a carboxy group, a keto group, an aldehyde group or ahemiacetale group may be mentioned. The keto group or the aldehyde groupor the hemiacetal group may be produced by chemically or enzymaticallyoxidizing a saccharide unit of the protein such as a saccharide unit ofa carbohydrate side chain of a glycosylated protein.

Depending on the chemical nature of the functional group of the protein,the functional group of the suitable compound which is reacted with thefunctional group of the protein is, e.g.:

-   -   C—C-double bonds or C—C-triple bonds or aromatic C—C-bonds;    -   the thio group or the hydroxy groups;    -   alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;    -   1,2-dioles;        -   1,2 amino-thioalcohols;        -   azides;    -   1,2-aminoalcohols;    -   the amino group —NH₂ or derivatives of the amino groups        comprising the structure unit —NH— such as aminoalkyl groups,        aminoaryl group, aminoaralkyl groups, or alkarlyaminogroups;    -   the hydroxylamino group —O—NH₂, or derivatives of the        hydroxylamino group comprising the structure unit —O—NH—, such        as hydroxylalkylamino groups, hydroxylarylamino groups,        hydroxylaralkylamino groups, or hydroxalalkarylamino groups;    -   alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups,        or alkaryloxyamino groups, each comprising the structure unit        —NH—O—;    -   residues having a carbonyl group, -Q-C(=G)-M, wherein G is O or        S, and M is, for example,        -   —OH or —SH;        -   an alkoxy group, an aryloxy group, an aralkyloxy group, or            an alkaryloxy group;        -   an alkylthio group, an arylthio group, an aralkylthio group,            or an alkarylthio group;        -   an alkylcarbonyloxy group, an arylcarbonyloxy group, an            aralkylcarbonyloxy group, an alkarylcarbonyloxy group;        -   activated esters such as esters of hydroxylamines having            imid structure such as N-hydroxysuccinimide or having a            structure unit O—N where N is part of a heteroaryl compound            or, with G=O and Q absent, such as aryloxy compounds with a            substituted aryl residue such as pentafluorophenyl,            paranitrophenyl or trichlorophenyl;    -   wherein Q is absent or NH or a heteroatom such as S or O;        -   —NH—NH₂, or —NH—NH—;        -   —NO₂;        -   the nitril group;        -   carbonyl groups such as the aldehyde group or the keto            group;        -   the carboxy group;        -   the —N═C═O group or the —N═S group;    -   vinyl halide groups such as the vinyl iodide or the vinyl        bromide group or triflate;        -   —C≡C—H;        -   (C═NH₂Cl)—OAlkyl        -   groups —(C═O)—CH₂-Hal wherein Hal is Cl, Br, or I;        -   —CH═CH—SO₂—;        -   a disulfide group comprising the structure —S—S—;    -   the group

-   -   the group

The functional group of the suitable compound and the thioester group orthe group G may be separated by any suitable spacer. Among others, thespacer may be an optionally substituted, linear, branched and/or cyclichydrocarbon residue. Generally, the hydrocarbon residue has from 1 to60, preferably from 1 to 40, more preferably from 1 to 20, morepreferably from 2 to 10, more preferably from 2 to 6 and especiallypreferably from 2 to 4 carbon atoms. If heteroatoms are present, theseparating group comprises generally from 1 to 20, preferably from 1 to8 and especially preferably from 1 to 4 heteroatoms. The hydrocarbonresidue may comprise an optionally branched alkyl chain or an aryl groupor a cycloalkyl group having, e.g., from 5 to 7 carbon atoms, or be anaralkyl group, an alkaryl group where the alkyl part may be a linearand/or cyclic alkyl group.

In case the group G is chemically modified with a further compound whichcomprises a functional group which is reacted with G and anotherfunctional group which is a thioester group —S—R′, the functional groupwhich is reacted with G may be suitably selected from the group offunctional groups described above with regard to Q. The thioester groupand the group which is reacted with G may be separated by any suitablespacer. Among others, the spacer may be an optionally substituted,linear, branched and/or cyclic hydrocarbon residue. Generally, thehydrocarbon residue has from 1 to 60, preferably from 1 to 40, morepreferably from 1 to 20, more preferably from 2 to 10, more preferablyfrom 2 to 6 and especially preferably from 2 to 4 carbon atoms. Ifheteroatoms are present, the separating group comprises generally from 1to 20, preferably from 1 to 8 and especially preferably from 1 to 4heteroatoms. The hydrocarbon residue may comprise an optionally branchedalkyl chain or an aryl group or a cycloalkyl group having, e.g., from 5to 7 carbon atoms, or be an aralkyl group, an alkaryl group where thealkyl part may be a linear and/or cyclic alkyl group.

Preferably, the protein is recombinantly produced. This includesprokaryotic or eukaryotic host expression of exogenous DNA sequencesobtained by genomic or cDNA cloning or by DNA synthesis. The recombinantproduction of a protein is known in the art. In general, this includesthe transfection of host cells with an appropriate expression vector,the cultivation of the host cells under conditions which enable theproduction of the protein and the purification of the protein from thehost cells.

According to a still further preferred embodiment, an expression vectoris used which leads to a thioester functionalized protein, preferably aC-terminal polypeptide-thioester. In one embodiment, the thioesterfunctionalized protein is bound via a C-terminal thioester bond to anintein polypeptide, preferably to an intein affinity-tag fusionpolypeptide. Such thioester functionalized proteins are, for example,obtainable by fusing the protein of interest in frame with a, preferablymutant, intein-CBD polypeptide, for example in the context of thevectors of the commercially available IMPACT (TM)—CN system, like pCYBor pTYB (New England Biolabs, Frankfurt/Main, Germany). The thioesterfunctionalized protein bound via a thioester bond to the Intein (TFPI)preferably comprises an affinity tag fused to the C-terminus of theintein, more preferably the affinity tag can be a chitin-binding domain(CBD), a polyhistidine-tag, a Strep-tag or GST. In one embodiment, theTFPI is further bound via an affinity tag to a corresponding affinityresin, for example bound to a chitin-bead via a CBD fused to the intein(see, for example, Muir et al. (1998) Proc. Natl. Acad. Sci. USA95:6705-6710). In a preferred embodiment, the TFPI is purified on theaffinity resin under conditions leaving the thioester linkage intact(see Muir et al., above). The TFPI bound to the affinity resin can thenbe reacted with the alpha-X beta-amino functionalized HES, preferably toalpha-X beta-amino functionalized HES such as alpha-X beta-aminofunctionalized HES with a mean molecular weight of about 10 kD.

It is preferred that a thioester functionalized protein bound via athioester bond to an alkyl-, aryl- or aralkyl-residue (TFPA) begenerated, e.g. by reacting the TFPI with an alkylthiol, arylthiol oraralkylthiol. In a preferred embodiment, the alkylthiol is not a dithioland is selected from methyl-, ethyl-, propyl- or butylthiol; ethylthiolis particularly preferred. The alkylthiol may also be amercaptoalkylcarbonic acid or a mercaptoalkyl-sulfonic acid.2-mercaptoethansulfonic acid is particularly preferred. In anotherembodiment, the arylthiol is not a dithiol and can be, for example,thiophenol, 1-thio-2-nitrophenol, 2-thio-benzoic acid, 2-thiopyridine,4-thio-2-pyridine carboxylic acid or 4-thio-2-nitropyridin. The TFPA canbe isolated, as described, e.g., in US 2002/0151006A1 in Example 3 foran Ab1-SH3 ethyl-thioester, and can either be used directly for thereaction with the alpha-X beta-amino functionalized HES or can even bepurified further and then used for reaction or can even be stored andthen be used for the reaction later. A further example of a TFPA isshown in Iakovenko, A., et al., FEBS Letters 468 (2000) 155-158, where athioester functionalized Rab7ΔC6 was generated in section 2.2. (athioester with 2-mercaptoethansulfonic acid), purified and stored beforeuse for a final coupling step.

Therefore, the present invention also relates to a method as describedabove, wherein the active substance is a protein which was producedusing an expression vector leading to a thioester functionalizedprotein.

According to another aspect, the present invention also relates to aconjugate obtainable by a method as described above.

It is envisaged that the method according to the present invention is achemoselective method, i.e. depending on the alternatives describedabove, coupling of hydroxyalkyl starch or a derivative thereof,preferably hydroxyethyl starch or a derivative thereof, takes place atspecific sites of the active substance such as selectively at theN-terminus of the active substance in case the active substance has aN-terminal cysteine residue, as described above, or selectively at theC-terminus of the active substance in case the active substance has aC-terminal thioester group as described above, wherein the activesubstance is reacted with a thioester functionalized hydroxyalkyl starchderivative and with an alpha-thiol beta-amino functionalizedhydroxyalkyl starch derivative, respectively.

The term “selectively at a terminus” as used in the context of thepresent invention relates to a process according to which statisticallymore than 50%, preferably at least 55%, more preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and still more preferably atleast 95% such as 95%, 96%, 97%, 98%, or 99% of active substancemolecules are exclusively reacted via the respective terminus.

The present invention also comprises an embodiment in which the thiolgroup of the alpha-thiol beta-amino group is replaced by a groupaccording to the formula

Therefore, instead of a cystein, a histidin may be employed, preferablyan N-terminal cysteine or histidine.

In the methods for preparing a conjugate of the invention the conversionrate in the above described methods may be at least 50%, more preferredat least 70%, even more preferred at least 80% and in particular 95% oreven more, such as at least 98% or 99%.

According to yet another aspect, the present invention also relates to aconjugate as described above or a conjugate, obtainable by a method asdescribed above, for use in a method for the treatment of the human oranimal body.

The conjugates according to the invention may be at least 50% pure, evenmore preferred at least 70% pure, even more preferred at least 90%, inparticular at least 95% or at least 99% pure. In a most preferredembodiment, the conjugates may be 100% pure, i.e. there are no otherby-products present.

Therefore, according to another aspect, the present invention alsorelates to a composition which may comprise the conjugate(s) of theinvention, wherein the amount of the conjugate(s) may be at least 50wt-%, even more preferred at least 70 wt-%, even more preferred at least90 wt-%, in particular at least 95 wt.-% or at least 99 wt.-%. In a mostpreferred embodiment, the composition may consist of the conjugate(s),i.e. the amount of the conjugate(s) is 100 wt.-%.

Accordingly, the present invention also relates to a pharmaceuticalcomposition comprising a conjugate as described above or a conjugate,obtainable by a method as described above.

Moreover, the present invention also relates to a pharmaceuticalcomposition comprising a conjugate as described above or a conjugate,obtainable by a method as described above, said pharmaceuticalcomposition further comprising at least one pharmaceutically acceptablediluent, adjuvant, or carrier.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Examples Example 1 Synthesis of Functionalized HES Example 1.1 Synthesisof Amino-HES from Oxidized HES

5.122 g of HES (MW=14,500 D, DS=0.41, Supramol Parenteral Colloids GmbH,Rosbach-Rodheim, D), selectively oxidized at its reducing end accordingto DE 196 28 705 A1, were heated at 80° C. in vaccuo for 15.5 h anddissolved under nitrogen in 25 mL dry DMSO (Fluka, Sigma-Aldrich ChemieGmbH, Taufkirchen, D). To the resulting solution, 51.22 mmol1,4-diaminobutane (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D)were added. After incubation for 17 h at 40° C., the reaction mixturewas added to 150 mL of an ice-cold 1:1 mixture of ethanol (DAB,Sonnenberg, Braunschweig, D) and acetone (Carl Roth GmbH +Co. K G,Karlsruhe, D) (v/v), and incubated at −20° C. for 1 h. The precipitatedproduct was collected by centrifugation at 4° C., washed with 40 ml ofthe same mixture, re-dissolved in 80 mL water, dialysed for 40 h againstwater (SnakeSkin dialysis tubing, 3.5 kD cut off, Perbio SciencesDeutschland GmbH, Bonn, D), and lyophilized. The product was isolated in67% yield.

Example 1.2 Synthesis of H-Cys(S-tBu)-HES from Amino-HES of Example 1.1

86.3 mg Fmoc-Cys(S-tBu)-OH (Fluka, Sigma-Aldrich Chemie GmbH,Taufkirchen, D) and 45.9 mg 1-hydroxy-1H-benzotriazole (Aldrich,Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 2 mLN,N-dimethylformamide (Peptide synthesis grade, Biosolve, Valkenswaard,NL), and 40.7 μL N,N′-diisopropylcarbodiimide (Fluka, Sigma-AldrichChemie GmbH, Taufkirchen, D) were added.

After incubation at 21° C. for 30 min, 206 mg of amino-HES, synthesizedas described in example 1.1, were added. After shaking for 19 h at 22°C., the reaction mixture was added to 14 mL of ice-cold 2-propanol (CarlRoth GmbH, Karlsruhe, D). The precipitated product was collected bycentrifugation at 4° C., re-suspended in 8 ml 2-propanol and centrifugedas described. The precipitate was dissolved in 20 mL water, 20 mldichloromethane (Carl Roth GmbH, Karlsruhe, D) were added and themixture was vortexed. After centrifugation, the upper aqueous layer wasisolated, dialysed for 43 h against water (SnakeSkin dialysis tubing,3.510 cut off, Perbio Sciences Deutschland GmbH, Bonn, D) andlyophilized. Fmoc-Cys(S-tBu)-HES was isolated in 67% yield.

100.7 mg of Fmoc-Cys(S-tBu)-HES was dissolved in 1 mL of 20% piperidine(Acros Organics, Geel, B) in DMF (v/v). After shaking for 15 min at 22°C., the reaction mixture was added to 20 mL of tert-butyl methyl ether(Acros Organics, Geel, B). The precipitated product was collected bycentrifugation at 4° C., re-suspended in 10 ml tert-butyl methyl etherand centrifuged. After drying, the precipitate was dissolved in 10 mLwater and dialysed for 31 h against water (SnakeSkin dialysis tubing,3.5 kD cut off, Perbio Sciences Deutschland GmbH, Bonn, D) andlyophilized. H-Cys(S-tBu)-HES was isolated in 80% yield.

Example 1.3 Synthesis of Thioester-HES from Amino-HES of Example 1.1

29.3 mg Pentafluorophenyl S-benzyl thiosuccinate (Link technologies,Bellshill, UK) and 10.1 mg 1-hydroxy-1H-benzotriazole (Aldrich,Sigma-Aldrich Chemie GmbH, Taufkirchen, D) were dissolved in 1.5 mLN,N-dimethylformamide (Peptide synthesis grade, Biosolve, Valkenswaard,NL), and 75 mg amino-HES, synthesised as described in example 1.1, wereadded. After shaking for 15 h at 22° C., the reaction mixture was addedto 15 mL of tert-butyl methyl ether (Acros Organics, Geel, B). Theprecipitated product was collected by centrifugation at 4° C.,re-suspended in 8 ml tert-butyl methyl ether and centrifuged. Theprecipitate was dried in a stream of nitrogen.

Example 2 Synthesis of Protein-HES Conjugates by Chemical LigationExample 2.1 Synthesis of a Peptide-Thioeter A-HES Conjugate fromH-Cys(S-tBu)-HES and Peptide-Thioester A

To 5 μL of a 10 mg/mL solution of the peptide thioesterPeptide-Thioester A (GBF, Braunschweig, D, amino acid sequence:H-SPFGADTTVCCFNYSVRKLPQNHVKDYFYTSSK-thiopropionic acid ethyl ester;MW=3,920 g/mol) in a 1:1 mixture (v/v) of N,N-dimethylformamide (peptidesynthesis grade, Biosolve, Valkenswaard, NL) and 0.1 M sodium phosphatebuffer, pH 7.2, 5 μL of a 255 mg/mL solution of H-Cys(S-tBu)-HES,synthesized as described in example 1.2, and 5 μL of a 1.5 M catalystsolution in DMF were added at 22° C. As catalyst, either thiophenol(FIG. 1, Lane B) or benzyl mercaptan (FIG. 1, Lane C) were employed. Asa reaction control, 5 μL buffer instead of the catalyst solution wereadded to the mixture of HES-derivative and peptide (FIG. 1, Lane E).

The mixtures were incubated over night at room temperature and analysedby gel electrophoresis (FIG. 1).

Example 2.2 Synthesis of a GM-CSF Inhibitor Peptide-HES Conjugate fromThioester-HES and GM-CSF Inhibitor Peptide

To 10 μL of a 2.82 mg/mL solution of the N-terminal Cys-peptide GM-CSFInhibitor Peptide (54-78) (Bachem AG, Order-No. H-3438, Bubendorf, CH,Amino acid sequence:H-Cys-Leu-Gln-Thr-Arg-Leu-Glu-Leu-Tyr-Lys-Gln-Gly-Leu-Arg-Gly-Ser-Leu-Thr-Lys-Leu-Lys-Gly-Pro-Leu-Thr-OH;MW=2,816.4 g/mol) in 0.1 M sodium phosphate buffer, pH 7.2, containing150 mM sodium chloride and 5 mM EDTA, 32 μL of a 156 mg/mL solution ofThioester-HES, synthesized as described in example 1.3, in the samebuffer and 1.7 μL, of thiophenol (FIG. 2, Lane B) were added at 22° C.

The mixture was incubated over night at room temperature and analysed bygel electrophoresis (FIG. 2).

Example 2.3 Reaction Control—Incubation of EPO with Thioester-HES

To 19.2 μL of a 0.78 mg/mL solution of EPO (recombinantly produced EPOhaving the amino acid sequence of human EPO and essentially the samecharacteristics as the commercially available Erypo® (Ortho Biotech,Jansen-Cilag) or NeoRocormon® (Roche), cf. EP 0 148 605, EP 0 205 564,EP 0 411 678) in 10 mM sodium phosphate buffer, pH 7.2, containing 150mM sodium chloride, 1.7 μL of thiophenol and 5 μL of either a 10 or 50mg/mL solution of thioester-HES, synthesized as described in example1.3, in 0.1 M sodium phosphate buffer, pH 7.2, containing 150 mM sodiumchloride and 50 mm EDTA (FIG. 3, Lanes B and C) were added at 22° C. Asa reaction control, the same reaction without thioester-HES wasperformed (FIG. 3, Lane D).

The mixture was incubated over night at room temperature and analysed bygel electrophoresis.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for preparing a conjugate of an active substance andhydroxyalkyl starch, wherein the active substance and the hydroxyalkylstarch are covalently linked by a chemical residue having a structureaccording to formula

wherein Y is a heteroatom, selected from the group consisting of O andS, said method comprising (i) reacting a thioester group —(C═Y)—S—R′ ofa hydroxyalkyl starch derivative comprising said thioester group with analpha-X beta amino group

of an active substance derivative comprising said alpha-X beta aminogroup, or (ii) reacting a thioestergroup —(C═Y)—S—R′ of an activesubstance derivative comprising said thioester group with an alpha-Xbeta amino group

of a hydroxyalkyl starch derivative comprising said alpha-X beta aminogroup, wherein R′ is selected from the group consisting of hydrogen, anoptionally substituted, linear, cyclic or branched alkyl, aryl,heteroaryl, aralkyl, and heteroaralkyl group, wherein X is selected fromthe group consisting of SH and

and wherein the group —(C═Y) is derived from the thioester group—(C═Y)—S—R′ and the group HN—CH—CH₂—X is derived from the alpha-X betaamino group.
 2. The method as claimed in claim 1, wherein a thioesterfunctionalized hydroxyalkyl starch is reacted with an alpha-X beta-aminogroup of the active substance wherein the alpha-X beta-amino group iscomprised in a cysteine or histidine residue of the active substance. 3.The method as claimed in claim 2, said method comprising oxidizinghydroxyalkyl starch at its reducing end and (i) converting the oxidizedreducing end to an activated carboxylic acid derivative and reacting theactivated carboxylic acid derivative with a compound R′-SH; or (ii)reacting the oxidized reducing end with a carbodiimide and a thiol R′SHto give the thioester functionalized hydroxyalkyl starch.
 4. The methodas claimed in claim 1, said method comprising oxidizing hydroxyalkylstarch at its reducing end, reacting the oxidized reducing end with anat least bifunctional compound comprising two amino groups to give anamino functionalized hydroxyalkyl starch derivative, and reacting theamino group of the derivative with an at least bifunctional compoundcomprising at least one functional group which is reacted with the aminogroup of the derivative, and comprising at least one thioester group. 5.The method as claimed in claim 1, wherein a thioester functionalizedactive substance is reacted with an alpha-X beta-amino group of ahydroxyalkyl starch derivative, said method comprising oxidizinghydroxyalkyl starch at its reducing end and reacting the oxidizedreducing end with a functional group Z of a compound comprising, inaddition to Z, an alpha-X beta-amino group.
 6. The method as claimed inclaim 5, wherein the compound comprising Z and the alpha-X beta-aminogroup is 1,3-diamino-2-thio propane or 2,3-diamino-1-thio propane. 7.The method as claimed in claim 1, wherein a thioester functionalizedactive substance is reacted with an alpha-X beta-amino group of ahydroxyalkyl starch derivative, said method comprising oxidizinghydroxyalkyl starch at its reducing end, reacting the oxidized reducingend with a functional group Z of a compound comprising, in addition toZ, a further functional group W, to give a first hydroxyalkyl starchderivative, and reacting the functional group W of the firsthydroxyalkyl starch derivative with a functional group V of a compoundcomprising, in addition to V, an alpha-X beta-amino group, to give thealpha-X beta-amino functionalized hydroxyalkyl starch derivative.
 8. Themethod as claimed in claim 7, wherein the compound comprising Z and W isa diamino functionalized compound.
 9. The method as claimed in claim 7,wherein the compound comprising V and the alpha-X beta-amino is cysteineor a derivative thereof or histidine or a derivative thereof, V beingthe carboxy group or a reactive carboxy group.
 10. The method as claimedin claim 5, wherein the active substance is a small molecule drugcomprising a carboxy group, said method comprising (i) converting thecarboxy group to an acid chloride with a compound R′-SH viaalkylthio-dehalogenation, or (ii) reacting the carboxy group with acarbodiimide and a thiol R′SH to give the thioester functionalizedactive substance.
 11. The method as claimed in claim 5, wherein theactive substance is a peptide which was produced using a synthesis resinallowing for a thioester functionalized peptide.
 12. The method asclaimed in claim 5, wherein the active substance is a protein which wasproduced using an expression vector leading to a thioesterfunctionalized protein.
 13. A conjugate of an active substance andhydroxyalkyl starch, as obtainable by a method as claimed in claim 1.14. A conjugate of an active substance and hydroxyalkyl starch, whereinthe active substance and the hydroxyalkyl starch are linked by achemical moiety, having a structure according to formula

wherein Y is a heteroatom, selected from the group consisting of O andS, and X is selected from the group consisting of SH and

said conjugate having a structure according to formula

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the thioester group, and wherein AS′ is theresidue of the active substance or a derivative thereof which was linkedto the alpha-X beta-amino group, or a structure according to formula

wherein HAS′ is the residue of the hydroxyalkyl starch or derivativethereof which was linked to the alpha-X beta-amino group, and whereinAS′ is the residue of the active substance or a derivative thereof whichwas linked to the thioester group, and wherein the group —(C═Y) isderived from the thioester group —(C═Y)—S—R′ and the group HN—CH—CH₂—Xis derived from the alpha-X beta amino group.
 15. The conjugate asclaimed in claim 14 having a structure according to formula

or according to formula

or according to formula

wherein n=0 or 1, wherein L is an optionally substituted, linear, cyclicor branched alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl residuewith 2 to 10 carbon atoms in the respective alkyl residue, wherein R₁,R₂ and R₃ are independently hydrogen or a hydroxyalkyl group, andwherein HAS″ refers to the HAS molecule without the terminal saccharideunit at the reducing end.
 16. The conjugate as claimed in claim 14,wherein the active substance is selected from the group consisting ofproteins, peptides, and small molecule drugs.
 17. The conjugate asclaimed in claim 14, wherein the hydroxyalkyl starch is hydroxyethylstarch having a mean molecular weight of from 1 to 300 kD and a degreeof substitution from about 0.1 to about 0.8.
 18. An alpha-X beta-aminofunctionalized hydroxyalkyl starch derivative according to formula

or according to formula

or according to formula

wherein L is an optionally substituted, linear, cyclic or branchedalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl residue with 2 to 10carbon atoms in the respective alkyl residue, wherein HAS″ refers to theHAS molecule without the terminal saccharide unit at the reducing end,and wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, and wherein X is selected from the group consisting of SH and


19. A thioester functionalized hydroxyalkyl starch derivative accordingto formula

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, wherein HAS″ refers to the HAS molecule without the terminalsaccharide unit at the reducing end, and wherein S—R′ is anelectrophilic leaving group.
 20. A thioester functionalized hydroxyalkylstarch derivative according to formula

wherein R₁, R₂ and R₃ are independently hydrogen or a hydroxyalkylgroup, wherein HAS″ refers to the HAS molecule without the terminalsaccharide unit at the reducing end, and wherein S—R′ is anelectrophilic leaving group, wherein L₁ and L₂ are independently anoptionally substituted, linear, branched or cyclic hydrocarbon residue,optionally comprising at least one heteroatom, comprising an alkyl,aryl, aralkyl, heteroalkyl, or heteroaralkyl moiety, said residue havingfrom 1 to 60 carbon atoms, wherein D is a linkage formed by a functionalgroup F₂ linked to L₁, and a functional group F₃ linked to L₂, andwherein F₃ is capable of forming a chemical linkage with F₂.
 21. Theconjugate as claimed in claim 20, wherein L₁ and L₂ are independently—(CH₂)_(n)- with n=2, 3, 4, 5, 6, 7, 8, 9, or
 10. 22. The conjugate asclaimed in claim 20, wherein F₂ and F₃ independently are selected fromthe group consisting of a C—C-double bond or C—C-triple bond or aromaticC—C-bond; a thio group or a hydroxy group; an alkyl sulfonic acidhydrazide or an aryl sulfonic acid hydrazide; a 1,2-diol; a 1,2 aminothioalcohol; an azide; a 1,2-aminoalcohol; an amino group —NH₂ or aderivative of an amino group comprising the structure unit —NH—; ahydroxylamino group —O—NH₂, or derivative of a hydroxylamino groupcomprising the structure unit —O—NH—; an alkoxyamino group, aryloxyaminogroup, aralkyloxyamino group, or alkaryloxyamino group, each comprisingthe structure unit —NH—O—; a residue having a carbonyl group,-Q-C(═G)-M, wherein G is O or S, and M is OH or —SH; an alkoxy group, anaryloxy group, an aralkyloxy group, or an alkaryloxy group; an alkylthiogroup, an arylthio group, an aralkylthio group, or an alkarylthio group;an alkylcarbonyloxy group, an arylcarbonyloxy group, anaralkylcarbonyloxy group, or an alkarylcarbonyloxy group; an activatedester having imide structure or having a structure unit O—N where N ispart of a heteroaryl compound or, with G=O and Q absent, an aryloxycompound with a substituted aryl residue; wherein Q is absent or NH or aheteroatom such as S or O; —NH—NH₂, or —NH—NH—; —NO₂; a nitril group; acarbonyl group; a carboxy group; a —N═C═O group or a —N═C═S group; avinyl halide group; —C≡C—H; —(C═NH₂Cl)—OAlkyl a —(C═O)—CH₂-Hal groupwherein Hal is Cl, Br, or I; —CH═CH—SO₂—; a disulfide group comprisingthe structure —S—S—; the group

and the group


23. A method for the treatment of a human or animal body, comprisingadministering the conjugate of claim 13 to a human or animal in need oftreatment.
 24. A pharmaceutical composition comprising a conjugate asclaimed in claim
 13. 25. A pharmaceutical composition as claimed inclaim 24, further comprising at least one pharmaceutically acceptablediluent, adjuvant, or carrier.
 26. A composition comprising a conjugateof an active substance and hydroxyalkyl starch as claimed in claim 14.27. A composition comprising an alpha-X beta-amino functionalizedhydroxyalkyl starch derivative as claimed in claim
 18. 28. A compositioncomprising a functionalized hydroxyalkyl starch derivative as claimed inclaim
 19. 29. A composition comprising a functionalized hydroxyalkylstarch derivative as claimed in claim
 20. 30. A thioester functionalizedhydroxyalkyl starch derivative as claimed in claim 19, wherein theelectrophilic leaving group S—R′ is selected from the group consistingof substituted or unsubstituted thiophenol, thiopyridine, benzylmercaptane, ethanethiol, methanethiol, 2-mercaptoethansulfonic acid,2-mercaptoacetic acid, 2-mercaptoacetic acid methyl or ethyl ester,3-mercaptopropionic acid, 3-mercaptopropionic acid methyl or ethylester, 4-mercaptobutyric acid, and 4-mercaptobutyric acid methyl orethyl ester.
 31. A thioester functionalized hydroxyalkyl starchderivative as claimed in claim 20, wherein the electrophilic leavinggroup S—R′ is selected from the group consisting of substituted orunsubstituted thiophenol, thiopyridine, benzyl mercaptane, ethanethiol,methanethiol, 2-mercaptoethansulfonic acid, 2-mercaptoacetic acid,2-mercaptoacetic acid methyl or ethyl ester, 3-mercaptopropionic acid,3-mercaptopropionic acid methyl or ethyl ester, 4-mercaptobutyric acid,and 4-mercaptobutyric acid methyl or ethyl ester.